Fig. 2: Performance evaluation of MB-based LAM detection assay.

a Schematic representation for generation of MB complex and TMB reaction for LAM detection. b Characterization of different assay steps of MB surface via UV–vis spectra absorbance at 260 nm, 280 nm, 650 nm. TEM image of (c) MB, (d) SSD–Ab–MB and (e) H-apt–LAM–SSD–Ab–MB using Ab and apt labeled with AuNPs with sizes of 60 and 20 nm, respectively (inset shows the corresponding high-magnification TEM images). f Calibration curves of UV-vis spectra at different concentrations of LAM in 0.1 M PBS (pH 7.4) and mixed urine (n = 20) at 650 nm, respectively. The LAM concentrations detected ranged from 10⁻² to 10³pg/mL. g Specificity of the assay for LAM detection. Concentrations of 1.0 × 10²pg/mL for ESAT-6, IFN-γ, IL-2, TNF-α and 1.0 × 10⁻¹pg/mL LAM were utilized. h Selectivity of the assay for LAM detection. Concentrations of C. albicans, S. aureus, B. cereus, E. coli, S. enterica, P. putida, S. aeruginosa lysates are 1.0 × 10⁷pg/mL and 1.0 × 10²pg/mL M.tb lysate were utilized. i Specificity of the assay against cross-reactive. Concentration of 0.1 mg/mL for IgG, 3 mg/mL for glucose, 3 mg/mL for BSA, 0.3 mg/mL for albumin and 0.01 mg/mL for hemoglobin were utilized. j Specificity of the assay against endogenous interference. Concentration of 0.5 mg/mL for uric acid, 15 mg/mL for urea, 3 mg/mL for creatinine, 2.3 mg/mL for KCl, 1.26 mg/mL for NH₄Cl, 1.75 mg/mL for NaCl, 1.75 mg/mL for CaCl₂ were utilized. k Specificity of the assay against exogenous interference. Concentration of isoniazid, rifampicin and paracetamol were 30 µg/mL, while the ibuprofen was 5 µg/mL. A concentration of 1.0 × 10¹pg/mL LAM was used in all interference tests (i–k). All data points and error bars correspond to average values and standard deviation obtained from four independent measurements. Data are presented as means ± standard errors of the mean. In the graph, asterisks indicate statistical significance as follows: ∗p  < 0.05, ∗∗p  < 0.01, and ∗∗∗p  < 0.001. Statistical analysis was conducted using a paired t-test.