Foldable paper microfluidic device based on single iron site-containing hydrogel nanozyme for efficient glucose biosensing

Peroxidase-mimicking nanozymes have been extensively studied, but their real utilization is limited by their working conditions limited to acidic pH, which makes it difficult to couple with oxidative enzymes operating at near-neutral pH. Here, we have developed a foldable paper microfluidic device that enables a highly convenient cascade reaction between peroxidase-mimicking nanozymes and oxidative enzymes to visually identify the target molecules. A single-atom iron-site-containing poly-γ-glutamic acid/chitosan hydrogel nanoparticle (PGA-Fe/CS NP) was developed, as a model peroxidase-like nanozyme, via rapid ionic gelation. Glucose oxidase (GOx) was used as a model oxidative enzyme. The paper device was constructed by simple wax-printing, including two parts with a foldable configuration: the first is for glucose oxidation under neutral pH and the second is for H2O2 reduction to produce a colorimetric signal under acidic pH. By dropping the sample solution in the first part, the device allowed efficient glucose oxidation by the immobilized GOx at neutral pH. On merely folding the device, the produced H2O2 from the glucose oxidation easily transfers to the second part of the device where PGA-Fe/CS NP was immobilized, inducing the peroxidase-like catalytic reaction at acidic pH. Using the foldable paper microfluidic device, the target glucose was successfully determined with excellent selectivity, sensitivity, stability, and reliability, using an image acquired by a smartphone. Since any oxidative enzymes could be incorporated into the device with the peroxidase-like PGA-Fe/CS NP, we expect that the foldable paper microfluidic device, which conveniently allows the cascade reaction under different pH environments, would be highly beneficial for the detection of various clinically important target molecules, thus laying the groundwork for future biosensing applications of nanozymes in point-of-care testing environments.