Rapid visual detection for nitroreductase based on the copper ions-induced and NADH-mediated aggregation of...

Hypoxia refers to the lack of oxygen supply to cells or tissues. The overexpression of nitroreductase has been shown to be closely related to the degree of hypoxia, which leads to the level of nitroreductase (NTR) being used as an indicator of hypoxia. We reported a facile visual detection of NTR based on the aggregation of gold and silver alloy nanoparticles. Compared with gold nanoparticles (AuNPs), the aggregation behavior of Au₈₀Ag₂₀ NPs caused a more prominent color change. Copper ions (Cu²⁺) can be rapidly reduced by nicotinamide adenine dinucleotide (NADH) under the catalysis of Au₈₀Ag₂₀ NPs. But NADH is consumed as an electron donor during the catalytic reduction reaction of p-nitrophenol (pNP) by NTR. A decrease of NADH amount results in the aggregation of Au₈₀Ag₂₀ NPs by the excess Cu²⁺ and different aggregation degrees of Au₈₀Ag₂₀ NPs lead to observable color change. A linear correlation of A₆₀₀/A₅₀₅ = 0.0285 [NTR]+0.361 (R² = 0.980) was obtained with a limit of detection (LOD) of 0.23 μg/mL for UV-vis spectrophotometer. For visual detection, the values of R/B against the concentration of NTR obtains a calibration curve of R/B = -0.031 [NTR]+ 1.54 (R² = 0.985) with a LOD of 0.76 μg/mL, which is of the same order of magnitude as the UV-vis spectrophotometer analysis. As a comparison, Au₈₀Ag₂₀ NPs was replaced by several different composition nanoparticles (Au NPs, Au₇₀Ag₃₀ NPs, Au₅₀Ag₅₀ NPs) to be a chromogenic substrate, and the results suggest the Au₈₀Ag₂₀ NPs is the most sensitive substrate in our assay. Selectivity tests showed that the detection system did not respond to other common substances, and the reaction mechanism was verified by inhibitor research. Finally, the assay was used on the human serum samples with spiking NTR, and the recovery rates of this assay with UV-vis spectrophotometer were basically consistent with RGB analysis.