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

Abstract

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) (K₃[Fe(CN)₆]). It is found that once K₃[Fe(CN)₆] is reduced to K₄[Fe(CN)₆], 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^-1 was achieved. Moreover, by adapting ALP as the enzyme label, an ECJP system-based colorimetric immunoassay protocol was established for sensitive detection of aflatoxin B₁ (AFB₁), and a detection limit as low as 0.73 pg mL^-1 was achieved. The developed immunoassay method is successfully applied to the detection of AFB₁ 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.

Keywords: Aflatoxin B(1); Colorimetric immunoassay; Just-in-time production; Oxidase-mimicking nanozyme.