(Invited) Fieldable Optical Biosensors with Integrated Sample Processing for Universal Surveillance and Diagnostics – Application to COVID-19

Abstract

There is an urgent need for broad-based surveillance and diagnostic strategies that can be applied quickly and effectively to address emerging pathogens, and preferably at the point of need, as is evident with the current covid-19 crisis. Our innate immune system is an example of such a sensor – a recognition system that is capable of identifying all pathogens quickly and sensitively. Conserved pathogen associated molecular patterns (PAMPs) produced by the infectious agents are recognized by immune receptors (e.g. Toll like Receptors, TLRs) in an exquisite scheme of pattern recognition, culminating in universal early identification pipeline. Mimicking innate immune recognition in the laboratory can therefore provide a strategy for universal diagnosis of infection, and this has been the focus of our research for over a decade.There are three objectives to be addressed in order to develop a universal diagnostic platform based on innate immune recognition. They are: 1) understanding host-pathogen biology, and the development of tailored assays to address biochemically disparate pathogen associated molecular patterns in complex clinical samples, 2) development of multiplex detection assays and ultra-sensitive sensor platforms for their detection, 3) field adaptation of sensors and sample processing and 4) validation in blinded clinical samples. Herein, we present the strategy and progress in each of these areas of development, as outlined below.In bacterial pathogens, pathogen signatures recognized by our innate immune receptors are typically amphiphilic in biochemistry – lipoproteins or glycolipids. These lipidic molecules are intrinsically unstable in the aqueous milieu of blood, making them difficult to detect. Work from our team lead to the understanding that all bacterial amphiphilic pathogen associated molecular patterns associate with host lipoprotein carriers, which function as a biological taxi service for their transport in blood. Understanding this, we developed tailored sample processing pipeline and assays (membrane insertion and lipoprotein capture) for the detection of amphiphiles in serum samples.In viral pathogens, the pathogen signatures recognized by our innate immune receptors are nucleic acids – DNA and RNA. We developed a bioinformatic pipeline – FEVER- to capture the conserved aspects of these signatures and develop our recognition probes to target these pathogens, and then developed amplification-free assays for their detection using molecular beacon technology.Assays developed for both bacterial and viral signatures were applied to a novel, fieldable waveguide-based optical biosensor platform developed by our team. This platform uses single mode planar optical waveguides for bio-detection within an evanescent field, offering much greater sensitivity (10X or higher) than conventional immunoassay platforms. This fieldable sensor (<8Lbs) with integrated phone based controls allows for bio-detection in the field, as is required for surveillance technologies. We have developed a multiplex detection pipeline on this platform using photostable and tunable quantum dots as the fluorescence reporters – allowing for the simultaneous discriminatory detection of said signatures. This novel patented approach allows for the use of conventional platforms, in addition to our biosensor for such interrogations. For adaptation of the method to field detection, we developed the first ever microfluidic lipid and nucleic acid separation system – which allows for 5 min disposable separation of signatures from blood in the field. The performance of this platform has been validated for the detection of biomarkers not only in laboratory samples, but also in complex clinical samples from high-disease burden clinical cohorts for a variety of diseases such as COVID-19, tuberculosis, bacterial sepsis, food-borne diseases as well as biosecurity and national security applications. Figure: Schematic representing the fieldable universal diagnostic platform and approach. Blood is a universal sample – where all pathogens are recognized by innate immune receptors. Blood is collected and processed on site using a microfluidics sample processing cassette for biomarker extraction. The presence of the biomarker is then measured using novel tailored assays on a fieldable portable waveguide biosensor offering an answer within 15 min. Figure 1