Diagnosing Tuberculosis By Quantification of Early Secretory Antigenic Target-6 (ESAT-6) in Dermal Interstitial Fluid (ISF) Using Microneedle Patches

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). TB is a global health problem that resulted in a total of 1.5 million death worldwide in 2020 and early diagnosis of the disease is crucial for a timely treatment. TB can exist in two forms, latent and active; latent disease does not require treatment whereas active disease does. However, there is currently no diagnostic tool that can differentially diagnose latent versus active TB at once.

Recently, microneedle patches (MNPs), which are arrays of micron-size needles, have been used as novel platforms for bodily fluid sampling and diagnosis in a minimally invasive and effective manner. Mainly, MNPs have been used to sample interstitial fluid (ISF), which is a relatively unexplored body fluid that fills in the space between cells in tissues, such as skin. As a rich source of biomarkers including proteins, metabolites, and nucleic acids that can be used for disease diagnosis, ISF is becoming a promising alternative to the conventional sources of biomarkers such as blood, urine, and saliva. Specific biomarkers in ISF can be directly captured by biorecognition elements (i.e., antibody) coated on the MNPs and can be further quantified via on-needle immunoassays such as enzyme-linked immunosorbent assay (ELISA).

The biomarker of interest in this study is Early Secretory Antigenic Target-6 (ESAT-6), a virulent factor secreted by metabolically active Mtb, which is useful for diagnosing active TB. Based on our preliminary study with collaborators at University of Georgia, we were able to quantify ESAT-6 concentration in both serum and dermal ISF of guinea pigs infected with Mtb at 6-weeks post infection (data unpublished). To determine the conditions necessary for in vivo applications of MNPs in capturing ESAT-6 and thereby diagnosing TB, it is pivotal to first optimize conditions for in vitro plate ELISA. This study focused on developing an in vitro plate ELISA for detecting ESAT-6 by varying numerous factors such as capture and detection antibody concentration, type of blocking buffer, incubation time and temperature. We developed in vitro ESAT-6 ELISA using a plasmonic-fluor reporter, with a detection limit of 0.27 pg/ml.