Smartphone-based point-of-care testing of isoniazid using an oxidase-mimicking Cu-N/C single-atom nanozyme

An oxidase-mimicking Cu-N/C single-atom nanozyme is developed for colorimetric detection of isoniazid (INH). • A H 2 O 2 -free “signal-off” sensing platform is established, enabling simplified operation and enhanced robustness. • A low detection limit of 0.16 μM is achieved, with successful validation in real-world samples and excellent agreement with HPLC. • Integration with a paper-based chip and a smartphone enables on-site INH detection. A colorimetric sensing platform based on Cu-N/C single-atom nanozymes (Cu-N/C SAzyme) was developed for the sensitive and selective detection of isoniazid (INH), a first-line anti-tuberculosis drug. The Cu-N/C SAzyme exhibits intrinsic oxidase-mimicking activity, generating hydroxyl radicals (⋅OH) and superoxide anion radicals (O 2 · − ) to synergistically oxidize colorless 3,3′,5,5′-tetramethylbenzidine (TMB) into its blue oxidized form (oxTMB) without the addition of H₂O₂. Upon introduction of INH, its strong reducing property rapidly reduces oxTMB and scavenges ·OH and O 2 · − radicals, leading to a concentration-dependent decrease in absorbance at 652 nm. The change in absorbance shows a good linear relationship with INH concentration over the range of 1–50 μM, with a detection limit of 0.16 μM. The method exhibits high reproducibility (RSD < 5%) and excellent selectivity, and has been applied to determine INH in pharmaceutical tablets and human serum samples, achieving recoveries of 95.1–104.7%. The proposed method shows good agreement with the standard HPLC method. Furthermore, a portable point-of-care testing (POCT) platform was established by integrating the Cu-N/C SAzyme with a paper-based chip and smartphone. This platform achieved a detection limit of 0.65 μM over the same linear range (1–50 μM), demonstrating its potential for on-site INH screening. Download: Download high-res image (146KB) Download: Download full-size image