One probe, two pathogens assay. Use of a single Au-nanoprobe for sample characterization harbouring a single target (M. tuberculosis or P. falciparum) and both targets. The amount of total DNA in each sample is constant (60 µg/mL). Au-nanoprobes aggregation was measured by ratio of aggregation (ratio of SPR intensity at 525 and 600 nm) for the assay mixtures—2.5 nM Au-nanoprobe, 10 mM phosphate buffer (pH 8) + 0.1 M NaCl, purified dsDNA targets (PCR products) at a final concentration of 60 ng/µL. All spectrophotometric data was collected 30 min after salt addition and error bars represent the standard deviation of three independent assays. The horizontal line represents the threshold of 1 considered for discrimination between positive (rAbs ≥ 1) and negative (rAbs < 1) result. A representative colorimetric results is showed upon each result bar-red positive result; blue/purple negative result.