Enzyme-controllable just-in-time production system of copper hexacyanoferrate nanoparticles with oxidase-mimicking activity for highly sensitive colorimetric immunoassay.

Nanozymes are a series of elaborately designed nanomaterials that can mimic the catalytic sites of natural enzymes for reactions. Bypassing the tedious design and preparation of nanomaterial, in this work, we report on a novel just-in-time production system of copper hexacyanoferrate nanoparticles (CHNPs), which act as an oxidase-mimicking nanozyme. This system can rapidly produce CHNPs nanozyme on demand by simply mixing Cu(II) with potassium hexacyanoferrate(III) (K[Fe(CN)]). It is found that once K[Fe(CN)] is reduced to K[Fe(CN)], the formation of CHNPs is inhibited. Therefore, the just-in-time production system of CHNPs was coupled with alkaline phosphatase (ALP) to construct an enzyme-controllable just-in-time production (ECJP) system, in which ALP could inhibit the production of by catalyzing the hydrolysis of ascorbic acid 2-phosphate (AAP) to generating ascorbic acid (AA). The ECJP system is then used to probe the activity of ALP by employing 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) as the chromogenic substrate, and a detection limit of 0.003 U L was achieved. Moreover, by adapting ALP as the enzyme label, an ECJP system-based colorimetric immunoassay protocol was established for sensitive detection of aflatoxin B (AFB), and a detection limit as low as 0.73 pg mL was achieved. The developed immunoassay method is successfully applied to the detection of AFB in peanut samples. The operation of ECJP system is quite simple and the coupling of ALP with CHNPs nanozyme can arouse dual enzyme-like cascade signal amplification. So, we believe this work can offer a new perspective for the development of nanozymes-based biodetection methods and colorimetric immunoassay strategies.