Understanding Cholesterol–Mycolic Acid–Phosphatidylcholine Interactions: Advancing Electrochemical Detection of Tuberculosis

Immunodetection of active tuberculosis (TB), including in human immnodeficiency virus (HIV)-positive patients, is crucial for effective treatment and disease elimination. Mycolic acid (MA) is a key antigen for detecting TB antibodies, although antimycolic acid antibodies (AMAAs) have not yet been isolated. However, AMAA levels are elevated in TB-infected patients and can interact with mycolic acid antigen (MAA). A significant challenge in TB detection arises from the cross-reactivity of cholesterol (Ch) and anticholesterol antibodies (AChAs) due to the cholesteroid nature of MAA. For the first time, the cholesteroidal nature of MAA has been established through electrochemical experiments and supported by theoretical density functional theory (DFT) calculations. An electrochemical TB immunosensor was developed by using a glassy carbon electrode modified with MAA-confined activated carbon (GCE-AC-MAA). Electrochemical analysis of TB-positive serum revealed activity similar to that of AChA in the presence of phosphatidylcholine (PC)/MAA, demonstrating cross-reactivity. The optimal detection protocol involved preincubating TB serum in liposomes to free AMAA, followed by electrochemical immunosensor detection. DFT calculations showed that cholesterol interacts with MAA (p-band center, ε_p = -11.3922 eV) but more strongly in the presence of PC/MAA (ε_p = -11.2695 eV). As the p-band center approaches the Fermi level, the bond length between cholesterol and the adsorbent shortens, increasing the interaction strength. The results indicate that the shorter the bond length between the adsorbate (Ch) and the adsorbent (PC, MA, or PC/MA), the stronger the p-band center (i.e., strong binding to the atomic nucleus). These findings provide valuable insights for improving TB immunodetection strategies.