Colorimetric and Photothermal Dual-Mode Apta-Sensor for Selective Detection of Kanamycin Using Chitosan-Stabilized Platinum Nanozymes

Addressing bacterial infections, kanamycin (KAN) finds extensive use in aquaculture, livestock husbandry, and agriculture. Nevertheless, the overuse of KAN leads to an excess of biological residues in the environment and food, resulting in potential nephrotoxicity, ototoxicity, and other harmful side effects for humans. Consequently, it becomes imperative to develop a rapid, selective, cost-effective, and sensitive detection system for KAN to safeguard human health and ensure food safety.<br/>Colorimetric assays employing antibodies have garnered significant attention due to their noticeable color change discernible by the naked eye, swift observation, cost-effectiveness, and ease of operation. Nonetheless, antibodies come with drawbacks such as high cost, sensitivity to storage conditions, and variability in quality from batch to batch, leading to decreased reproducibility. To address these challenges, we opted for aptamer molecules. These functional, single-stranded nucleic acids possess the ability to fold into specific 3-D structures, exhibiting high specificity for target molecules, whether they are small molecules, or even proteins.<br/>Nanozymes, nanomaterials exhibiting natural enzyme-like activities, have been recognized as viable substitutes to emulate natural enzymes such as horseradish peroxidase (HRP). This is attributed to their advantages of low cost, straightforward preparation, easy modification, excellent stability, and remarkable catalytic activity. Platinum nanoparticles (PtNPs), in particular, have found extensive use in bioanalytical applications due to their superior peroxidase-like activity, biocompatibility, and high chemical inertness. The choice of suitable stabilizing reagents is crucial in the synthesis of efficient nanozymes, as stabilizers significantly impact the physicochemical and biological properties of nanoparticles. In our study, chitosan (CS) was employed. The positively charged nature of CS facilitates the electrostatic binding of the negatively charged KAN binding aptamer (KBA) to chitosan-stabilized platinum nanoparticles (CS/PtNPs).<br/>Traditionally, immunoassays operate by detecting the target in a single mode. However, outcomes obtained through a single-mode signal readout can be susceptible to variations stemming from differences among operators, equipment disparities, and non-standard analytical protocols. In contrast, dual-mode sensing platforms not only possess the intrinsic characteristics of each response mode but also offer mutual validation of detection results obtained through diverse modes. Recent efforts have been directed towards establishing analytical methods that incorporate temperature changes as a signal, in addition to the colorimetric method, in immunoassays. Among various photothermal agents, oxidized TMB (TMB_ox) has emerged as a promising photothermal sensor due to its high photoconversion efficiency in near-infrared (NIR)-induced photothermal applications. TMB_ox exhibits distinct absorbance at UV-visible (652 nm) and NIR (808 nm) wavelengths, making it suitable for use as a probe in both colorimetric and photothermal detection.<br/>In this work, CS/PtNPs and TMB_ox were used to construct a dual-mode colorimetric and photothermal aptasensor designed for the selective detection of KAN, relying on the KBA-derived enhancement of peroxidase activity in CS/PtNPs and its subsequent inhibition by KAN. The developed dual-mode colorimetric and photothermal aptasensor exhibited a linear detection range of 0.1–50 and 0.5–50 μM, with respective limits of detection (LOD) of 0.04 and 0.41 μM. Furthermore, the aptasensor effectively identified KAN concentrations in spiked milk samples, confirming its reproducibility and reliability in practical applications. Crucially, the proposed aptasensor eliminates the need for a washing step and demonstrates robust selectivity for KAN. These characteristics position the aptasensor as a versatile tool for the detection of small molecules with known aptamers.