The goal of this proposal is to greatly improve the detection sensitivity and reduce the cost of biosensors for detecting biological entities. Genetically engineered yeast strains will be developed to enable highly specific detection while maximizing sensitivity. Application to clinically relevant proteins that are below the current detection limit will be investigated. Application of the biosensors are expected in the fields of food safety, biological security, monitoring environmental pollution, and detecting disease indicators.<br/><br/>The goal of the work is to develop a cost-effective technology that enables extremely high sensitivity immunosensing by applying genetically engineered cells as an immunoaffinity reagent and signal amplification. The approach will be tested for detecting a model biomarker in the diagnosis early indication of neurodegeneration. Genetic engineering approaches allow generation of cells with pre-designed biological functionality which can be mass-produced at low cost. In this work, specifically designed yeast strains that "capture" a target antigen will be made, and others that "detect/amplify" the target binding signal will also be incorporated. Since the engineered cells contain thousands of both reporter (e.g. fluorescent protein) and/or immunosensing entity (e.g. antibody fragment), multilevel signal amplification after immunoreaction can be realized, which facilitates the detection of ultra-low concentration. To ensure specificity of detection, control cells expressing a distinct reporter and control antibody fragment (not binding to the target) will be included in each sample, enabling ratiometric quantitation of specific signal over non-specific background. Since both "capture" and "detection/amplification" reagents can be produced by culturing modified yeasts, the immunoassay cost can potentially be reduced. The major research objectives are: generation and characterization of engineered "capture" and "detection/amplification" yeast cells; optimization of expression level and accessibility for maximizing detection sensitivity; and finally, application of the novel multifunctional signal amplifiers for immunoassay of human Tau protein (an Alzheimer's disease biomarker). The proposed genetically engineered multifunctional signal amplifiers are not limited to the proposed specific application, and can be generalized as a universal platform for monitoring any biomolecule of interest. The work also integrates research aims with educational activities, via the development of a new graduate course and undergraduate research.