Colorimetric Detection of Mycobacterium tuberculosis ESX‑1 Substrate Protein in Clinical Samples Using Au@Pd Nanoparticle-Based Magnetic Enzyme-Linked Immunosorbent Assay

The development of sensitive and point-of-care diagnostic tools for the serological profile of numerous Mycobacterium tuberculosis (MTB) rectifies the existing flaws in tuberculosis diagnosis. Herein, an ultrasensitive antigen-based tuberculosis detection technique and a bacterial growth monitoring strategy were designed using two nanozyme probe-based colorimetric enzyme-linked immunosorbent assay (ELISA). An anticulture filtrate protein-10 (CFP-10) factionalized magnetic nanobead (MNB) probe segregated the CFP-10 antigens from clinical samples, whereas the Au core and Pd shell (Au@Pd) nanozyme detection probe catalytically oxidized the commercially available chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB) to yield a concentration-dependent color tonality as a signal indicator. The developed MNB–Au@Pd NZ sandwich ELISA monitors an early bacterial growth using CFP-10 antigens secreted from MTB into the liquid culture, where the limit of detection reached as low as 5.6 × 10–12 g mL–1 with a linear tonal range between 5.0 × 10–13 and 5.0 × 10–4 g mL–1. Furthermore, active tuberculosis was discriminated from clinically isolated samples of human serum (40 samples) and urine (28 samples), with an accuracy of 92% (sensitivity: 93%; specificity: 91%) and 92% (sensitivity: 92%; specificity: 95%), respectively. This NZ-based colorimetric ELISA presents a robust and sensitive analytical technique for antigen-based clinical diagnosis, with potential applications in the clinical field, as well as for other epidemic diseases.