ABSTRACT Infections with Treponema pallidum, the causative agent of syphilis, have been rapidly rising in the United States over the past decade and present an increasing threat to public health. However, currently available diagnostic methods for T. pallidum infection remain suboptimal: they are time consuming, costly, are variably sensitive and specific for disease, are subject to false positive and false negative results, and are especially complicated to apply for the diagnosis of congenital syphilis. Moreover, most available testing algorithms require involvement of clinical reference laboratories able to support high complexity testing, increasing the overhead surrounding testing and the time necessary to achieve a positive or negative diagnostic result. As a result of both the practical challenges of testing and the inherent diagnostic limitations of available tests, some diagnoses are missed, and others are not made in a timely fashion, such that patients may be lost to follow-up before treatment. There is an increasingly urgent need for robust, rapid, low cost, and widely accessible testing to enable the diagnosis of T. pallidum in adult and congenital contexts. In response to these needs, we propose a new diagnostic paradigm based on the detection of T. pallidum antigens using aptamers, short ssDNA molecules that bind to target molecules with equal or greater affinity than antibodies. This innovative approach will enable ultrasensitive, point-of-care (POC) testing for T. pallidum infections from a variety of specimen types, such that syphilis can be rapidly diagnosed in adults and babies and appropriate therapy administered in real time. In our first Aim, we will develop aptamers that strongly and specifically bind T. pallidum proteins having high value as diagnostic targets. We will then evaluate the performance characteristics of those aptamers by measuring their sensitivity of binding to cultured T. pallidum strains, and their specificity of binding by confirming a lack of binding to related treponemal organisms, other genitourinary pathogens, and organisms from the normal human microbiota. In our second Aim, we will use these aptamers to develop methods and protocols for the rapid and ultrasensitive detection of T. pallidum antigens from clinical specimens. Our strategy will utilize polymerase-assisted cyclic signal amplification of organism-specific aptamers to generate and amplify fluorescent signal that can be detected at POC using inexpensive instrumentation. After protocol optimization, we will perform initial clinical validation of the assay using residual human specimens spiked with cultured T. pallidum. Our overall goal is to generate an inexpensive, ultrasensitive, and rapid detection method for syphilis that can be implemented at POC, so that accurate diagnosis of syphilis and administration of effective therapy can be implemented in the same healthcare encounter.