(447h) Ammonia Plasma Modified NiO Nanozymes for Colorimetric Biosensors

Monitoring of uric acid (UA) as biomarker for gout, high blood pressure, heart diseases, and kidney diseases is generally achieved by oxidizing a colorimetric reagent, such as 3, 3′, 5, 5′-tetramethylbenzidine (TMB), with H₂O₂ in the presence of catalyst such as the enzyme peroxidase. This reaction is inhibited by the presence of UA, allowing the concentration of UA to be determined based on the color change of the solution. The key factor to consider is that natural enzymes need precise operating conditions to operate effectively in biosensing platforms. To address this challenge, various techniques have been explored for synthesizing nanozymes. Although nanozymes have great potential, but they are not reusable. In addition to this, nanozymes tend to have a low density of active sites which result in lesser catalytic activity than natural enzymes. We propose an advanced biosensor for the natural enzyme-free colorimetric detection of UA, using a nanostructured NiO film as a peroxidase mimic to accelerate the oxidation reaction of colorless TMB to a blue product (TMBox) mediated by H₂O₂.

In this work, we fabricated a reusable NiO glancing angle deposited (GLAD) thin films on Si substrate which can mimic horseradish peroxidase (HRP, natural enzyme) for accelerating the oxidation reaction of TMB to oxTMB. GLAD is physical vapor deposition method which results highly porous and abundant active sites in thin films. As these thin films were surface-anchored structures, they could be easily recovered. Calculated Michaelis Menten constant (K_m) of the Ni GLAD film ranged ~1 mM, which confirmed high peroxidase-like activity but not up to the mark of natural peroxidase enzyme (K_m = 0.43 mM). Therefore, we further enhanced the surface energy of NiO nanozymes using ammonia plasma, which resulted in very high catalytic-active centers in the form of adsorbed N^3- ions. These ions can facilitate electron transfer and led to increased catalytic activity, low UA LOD (1 µM), and improved K_m (0.17 mM). Moreover, the developed colorimetric sensor was also tested against glucose and urea. The potential application of functionalized GLAD nanozymes is also demonstrated through a gravity-driven continuous catalytic reaction device for colorimetric sensing of UA using paper based point of care device.