Innovative polymer-based electrochemical platform for detecting ESAT-6 in human blood for pulmonary tuberculosis diagnosis

Pulmonary tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), necessitates early diagnosis for effective patient care. Despite advancements in TB diagnostics, there remains an urgent need to discover innovative non-sputum-based methods to detect Mtb-specific antigens for TB patient identification. We have developed a polymer-based electrochemical biosensor for detecting an Mtb-specific antigen, the 6-kDa early secreted antigenic target (ESAT-6), in blood. Using a gold electrode (Au), the biosensor is created by electropolymerizing poly(3,4-ethylene dioxythiophene) with carboxyl groups (PEDOT-COOH), which is activated with 3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (EDC-NHS), conjugated with an ESAT-6 polyclonal antibody (Ab), treated with bovine serum albumin (BSA) to block non-specific binding, forming BSA/Ab-EDC-NHS/PEDOT-COOH/Au. Using differential pulse voltammetry measurements, the electrode demonstrated an excellent linear response (R2 = 0.99) for ESAT-6 detection across a concentration range of 24.2 pM (0.81 ng/mL) to 50 nM (1.69 μg/mL), with a low detection limit of 1.39 pM (0.047 ng/mL) and a rapid detection time of under 4 min. This biosensor for ESAT-6 detection effectively distinguished pulmonary TB patients from healthy individuals, achieving 95.0 % sensitivity and 100 % specificity at a cut-off value of 97.0 ng/mL. It demonstrated a diagnostic accuracy of 97.1 %, outperforming the 82.9 % achieved by a commercial ELISA kit. Moreover, biosensor-detected ESAT-6 levels were significantly higher in smear-positive TB patients compared to the smear-negative group (p = 0.014), whereas ELISA-based detection showed no significant difference (p = 0.197). In conclusion, the PEDOT-COOH biosensor enables rapid and effective detection of plasma ESAT-6, facilitates TB diagnosis, and correlates with Mtb bacterial burden, highlighting its potential for disease monitoring.