SBIR-STTR Award

Linking infectious agent diagnostics to clinical decision-making at the point-of-care, requires a fast, highlysensitive and simple to use method that takes into account the infrastructure and workflow in clinical settings ofboth developing and developed regions. In addition, being able to distinguish among multiple potentialinfectious pathogens in a single test is of great importance as many pathogens cause similar symptoms. Thegoal of this phase I proposal is to develop and validate a highly sensitive biosensor diagnostic platformallowing naked eye detection of viral antigens at ? 1pM concentration in human serum. Our initial focus willbe on antigens from hemorrhagic fever viruses including Ebola and Dengue that are classified as Category ANIAID priority pathogens. The proposed diagnostic platform integrates three key features i) capture and isolationof target vial antigens from a complex biological sample (e.g. blood) using magnetic and silica microbeadsfunctionalized with specific antibodies to Ebola glycoprotein, VP40 or Dengue non-structural protein 1 followedby elution of isolated silica beads away from antigen/antibody complex (?antigen to bead conversion?); ii)enrichment of isolated silica beads by targeted flow of elution solution onto a nanohole array sensor with anoptically thick gold film; iii) naked eye detection through visible light induced shift in surface plasmonicresonance from the nanohole array. The sensitivity of the nanohole sensor is based on two important scientificdiscoveries: (i) Extraordinary Light Transmission (EOT) effect, and (ii) highly dispersive plasmonic Fanoresonances that eliminates the need for powered operated light sources or optical detectors. Recombinant viralproteins spiked in commercially available human serum will be used to evaluate the sensitivity, specificity andreliability of this diagnostic platform. At the end of this phase I grant a working nanophotonic prototype willprovide the basis for advancing this novel viral diagnostic platform for phase II development.

Thesaurus Terms:
Accounting; Antibodies; Antigens; Base; Biological Assay; Biological Markers; Biosensor; Categories; Clinical; Clinical Decision-Making; Coin; Communicable Diseases; Cost; Data; Dengue; Design; Detection; Detector; Developing Countries; Development; Devices; Diagnosis; Diagnostic; Ebola Virus; Electromagnetics; Enzyme-Linked Immunosorbent Assay; Equipment; Eye; Foundations; Future; Goals; Grant; Hemorrhagic Fever Virus; Human; Improved; Individual; Infection; Infectious Agent; Knowledge; Label; Lateral; Light; Light Transmission; Link; Medical; Methods; Microspheres; Nanofabrication; Nanoimprinting; Nanophotonic; National Institute Of Allergy And Infectious Disease; Novel; Optics; Output; Pathogen; Patient Care; Patients; Phase; Plasmonics; Point Of Care; Point-Of-Care Diagnostics; Price; Process; Prototype; Pump; Recombinants; Research Infrastructure; Sensitivity And Specificity; Sensor; Serum; Simulation; Source; Staging; Surface; Symptoms; Syringes; Technology; Testing; Time; User-Friendly; Viral; Viral Antigens; Viral Proteins; Virus; Visual; Work;

Ideally, the utilization of point-of-care (POC) diagnostics to facilitate early clinical decision-making for infectious disease agents requires compatibility in both developing and developed settings. Herein, we propose to exploit a novel localized surface plasmon resonance (LSPR) digital sensing technology to provide low cost, simple to use (single-step, no wash step assay), rapid (15-20 minutes), highly sensitive (<1ng/mL), and quantitative diagnostics compatible with a diverse range of POC settings and infectious diseases. The metallic thin-film sensors are composed of alternating layers of nano-structured gold and dielectric that emits a highly reproducible and stable plasmon upon exposure to white light that confers a distinct color to the thin-film. When specific biomarkers are localized to the sensor surface they interfere with the plasmon resulting in corresponding color changes that are digitally-monitored with a simple camera-based optical reader using RGB and proprietary algorithms to enable both high-sensitivity and precise quantification of a target biomolecule in a sample. Furthermore, unique to the LSPR sensing platform is the exceedingly short sensing range (localized at and extending a few tens of nm from the surface) that enables a single-step assay format (no wash necessary), which considerably simplifies disposable cartridge design. In this proposal we detail plans to provide POC diagnostics for acute dengue and Zika virus infection. The sensors will be integrated into a small fluidic assay cartridge that utilizes simple passive capillary action to move sample to the sensing region to selectively detect flavivirus specific NS1 proteins from serum in a rapid 20 minute assay format. We further propose herein to provide a versatile, low cost and robust hand-held portable camera-based digital reader compatible across a diverse range of POC settings. The reader will contain blue-tooth and Wi-Fi functionality, and a user-friendly interface with proprietary software to detect and quantify the LSPR signal / viral NS1 antigen. Prototype diagnostics will be validated against dengue and Zika patient serum samples to determine sensitivity. Furthermore, the dengue and Zika patient samples as well as normal and other infectious disease serum samples will be used to test for cross-reactivity (i.e. specificity). The goal is to provide a working alpha-prototype that demonstrates ?90% sensitivity and specificity.

Public Health Relevance Statement:
A highly sensitive, specific, portable, quantitative, rapid and user-friendly diagnostics for acute dengue and Zika will enable differential diagnosis and potentially prognosis for severe dengue to improve patient care across diverse geographies and clinical settings.

Project Terms:
Acute; Adult; Algorithmic Software; Algorithms; Americas; Antibodies; Antigens; Area; assay development; Auras; base; Biological Assay; Blood capillaries; Blood specimen; Bluetooth; Cessation of life; Climate; Clinical; clinical decision-making; Color; Communicable Diseases; Computer software; cost; cross reactivity; Culicidae; Data; Dengue; Dengue Hemorrhagic Fever; Dengue Infection; Dengue Shock Syndrome; Dengue Virus; design; Detection; Development; Development Plans; Diagnosis; Diagnostic; diagnostic assay; Differential Diagnosis; digital; Disease; Disease Outbreaks; Dose; driving force; Electronics; Enzyme-Linked Immunosorbent Assay; Erythrocytes; Evaluation; Exhibits; Exposure to; Extravasation; Film; Filtration; Flavivirus; flu; Geography; Goals; Gold; Guillain-Barré Syndrome; Hand; Human; Hydration status; Immunological Diagnosis; improved; Infant; Infection; Lead; Light; light intensity; Liquid substance; Medical center; Medical Research; Membrane; metallicity; Microcephaly; Monitor; Motion; Nanostructures; Neurologic; novel; Optical Readers; Optics; outcome forecast; Patient Care; Patients; Performance; point of care; point-of-care diagnostics; Point-of-Care Systems; Population; portability; prognostic; prototype; Quantitative Reverse Transcriptase PCR; Reader; Reproducibility; response; Risk; sample collection; Sampling; Sensitivity and Specificity; sensor; sensor technology; Serotyping; Serum; Serum Proteins; Signal Transduction; specific biomarkers; Specificity; Surface; Surface Plasmon Resonance; Symptoms; System; Testing; Thinness; user-friendly; Validation; validation studies; Variant; Vector-transmitted infectious disease; Viral; Virus; Whole Blood; wireless fidelity; Zika Virus; ZIKV infection