Abstract Syphilis is a sexually transmitted infection caused by bacterial pathogen Treponema pallidum (T. pallidum). Alt- hough it can be effectively treated with a single dose of long-acting penicillin, syphilis is re-emerging as a global public health problem. According to the CDC, the cases of adult and congenital syphilis in the U.S. increased 74% and 203% from 2017 to 2021, respectively. Therefore, there is an urgent need for better preventive measures for syphilis. Currently, most syphilis cases are diagnosed based on serum antibody tests that have been used in some fashion since the 1930s. In addition to imperfect specificity and sensitivity, serum antibody tests are unable to reliably distinguish between current and past infections, leading to under diagnosis and over treatment. The overall objective of this project is to develop a one-step, rapid, and low-cost point-of-care (POC) diagnostic assay for active T. pallidum infection. This POC assay is predicated on the recently developed Nano2RED technology, an in-solution assay pipeline that transduces antigen binding signals into physical ag- gregation of binder-functionalized gold nanoparticles (AuNPs) in < 30 minutes with atto- to pico-molar sensitivity. In this study, we will first engineer specific T. pallidum antigen binders templated on designed ankyrin repeat protein (DARPin), a versatile low-cost antibody-mimic protein scaffold with high tolerance to harsh/denaturing conditions and long storage stability (Aim 1). Next, we will create DARPin-based Nano2RED (DARPin-RED) and determine its sensitivity and specificity against a panel of syphilis patient samples (Aim 2). Since DARPins can be easily engineered to bind diverse targets, the successful completion of this project should yield not only a novel syphilis diagnostic assay for active T. pallidum bacteremia, but also a plug-and-play assay platform for potentially many other infections and diseases.