Rapid visual detection for nitroreductase based on the copper ions-induced and NADH-mediated aggregation of gold-silver alloy nanoparticles

Abstract

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.