A multi-line platinum nanozyme-based lateral flow device for the colorimetric evaluation of total antioxidant capacity in different matrices

Abstract

The evaluation of Total Antioxidant Capacity (TAC), namely the complete pattern of antioxidant species in a complex medium, is of major interest in many fields ranging from health monitoring to quality control in the food industry. In this framework, point-of-care (POC) testing technologies are a promising diagnostic solution for rapid on-site analyses, unlike laboratory based-assays, which are often limited by centralized analyses, time-consuming and costly procedures, and invasiveness in the case of health diagnostics. In this work, we developed a POC methodology that evaluates TAC in different matrices, exploiting the peroxidase-like properties of 5 nm platinum nanoparticles (PtNPs), combined with a colorimetric paper-based device. Notably, we designed and optimized a multi-line PtNPs-based Lateral Flow Assay (LFA), which relies on three sequential test lines with increasing concentrations of platinum nanozymes, to get a non-invasive, accurate, and fast (10 minutes) colorimetric evaluation of the body TAC in saliva samples. Furthermore, we employed the device as a prototype of a quality control tool in the food industry, for the determination of the TAC in fruit juices.

Fig. 1. Design and detection mechanism of the Pt nanozyme-based LFA. (a) Scheme of the device, based on three test lines at increasing concentrations of PtNPs. The antioxidant species in saliva compete with the chromogenic probe at the detection zone, preventing the oxidation of TMB and providing the color change of a number of test lines inversely proportional to the amount of AOX molecules. H₂O₂ is added to the saliva sample in advance, leading to the formation of the hydroxyl radicals at the nanoparticles' surface and leading to the oxidation of the reduced species (b). Antioxidants compete with TMB primarily directly interacting with hydroxyl radicals, and secondarily by reversing the TMB oxidation reaction. (c) As a result, a sample with a high amount of antioxidants shows one single test line, corresponding to the higher concentration of PtNPs. A medium TAC sample presents two test lines, while a sample with a low antioxidants level shows all the three test lines, since the amount of antioxidants is too small to compete with TMB.

Fig. 2. Proof-of-concept experiment to determine antioxidant content in saliva samples. (a) Vials showing the visual results of the UV-vis spectrophotometry analysis performed on the considered samples. As illustrated, the intensity of the blue color increases while decreasing the antioxidant content, in agreement with the Abs₆₅₂ value on top of each vial. (b) Representative pictures of the three devices evaluated at 10 minutes. The number of lines increases while decreasing the antioxidant levels in the samples. “Saliva + GSH”: physiological saliva sample with an addition of 10 mM glutathione; “Saliva”: physiological saliva sample without further modifications; “Diluted saliva”: physiological saliva sample diluted with 90% of pure water.

Fig. 3. Body TAC assessment in real saliva samples. (a) The 9 samples evaluated were divided into three groups (based on the Abs₆₅₂ level), each one characterized by a different amount of antioxidant species (high, medium, and low AOX levels). The number of test lines is clearly visible on each device. (b) ImageJ analysis of the 9 samples reporting the average values of the red coordinate (complementary of the blue color) and the relative standard deviations corresponding to the first, second, and third test line (TL) of each group (samples with high, medium, and low TAC level, respectively). In the “low” group, the red value relative to the first test line has lower intensity because the concentration of PtNPs used was very small (4 ppm), in order to better discriminate the “medium” group from the “low” group. The dashed line serves as a reference to better highlight the noise threshold.

Fig. 4. Antioxidant levels evaluation in fruit juices. Representative pictures of the devices after been tested with the three fruit juice's samples. One, two and three blue test lines are respectively shown for high, medium and low AOX levels samples. Abs₆₅₂ levels are respectively reported for each sample: red orange (“High”), Abs₆₅₂ ≈ 0.2; peach (“Medium”), Abs₆₅₂ ≈ 0.6; apricot (“Low”), Abs₆₅₂ ≈ 1.