ABSTRACT Invasive fungal diseases (IFDs) represent a large and growing problem in the U.S. Conventional methods for detecting IFDs include blood culture and histopathology, which require two to three days to complete. As a precautionary measure, patients are often given broad-spectrum antibiotics, which are ineffective against IFDs and can damage patients’ commensal microbiota, leading to severe dysbiosis and related health issues. To address this issue, several serological assays have been proposed to detect antigens or antibody responses in blood. However, these methods have failed to supplant clinical mainstays due to high cost, complexity, and most of all, limited sensitivity and/or specificity, especially during the early phases of infection. Our central hypothesis is that development of a simple and sensitive point-of-care (POC) device that can capture and purify multiple biomarkers simultaneously will radically improve the value of serodiagnoses. Our broad objective is to develop a handheld acoustic pipette for the rapid and efficient isolation of fungal biomarkers and prepare those biomarkers for downstream analysis. The outcome of this exploratory research will be a versatile prototype for multiplexed serological detections that is amenable to automation. This instrument will be distinguished from other IFD biosensing technologies through its innovative use of negative acoustic contrast particles (NACPs). Due to their elasticity, NACPs can immobilize against the pressure antinodes of an acoustic standing wave. This will allow us to capture specific biomarkers (e.g., antibodies, antigens), acoustically trap them within the device, and flush blood debris as waste. Then, captured biomarkers will be mixed and labeled with secondary fluorescent antibodies using microstructured fluidic chambers in a disposable cartridge housed within the pipette. Once biomarkers have been washed and stained, NACPs will be ejected from the device for analysis by standard analytical techniques, like microscopy or flow cytometry. We focus on C. albicans as a model IFD pathogen in this proposal due to its unique and well-characterized biomarkers. We propose three Specific Aims. Aim 1 will develop an acoustic pipette for rapid NACP mixing, trapping and washing. Aim 2 will determine the assay sensitivity and specificity for detecting C. albicans biomarkers: anti-Eno antibodies, anti- Fba1 antibodies and Eno antigens. Aim 3 will develop a multiplexed assay to simultaneously isolate and distinguish multiple biomarkers for C. albicans using barcoded NACPs. Aim 3 will also extend this multiplexing concept to screen for L. prolificans, a fungus that is fully resistant to amphotericin B, to inform early treatment decisions for antifungal medications. If successful, this technology will dramatically shorten the time needed to isolate and detect biomarkers that may impact not only the diagnosis of candidiasis, but also more lethal and obscure IFDs in an ergonomic and semi-a...