This work presents a simple hydrothermal synthesis of nitrogen-doped carbon dots (N-CDs), fabrication of microfluidic paper-based analytical device (μPAD), and their joint application for colorimetric determination of total cholesterol (TC) in human blood. The N-CDs were characterized by various techniques including transmission electron microscopy (TEM), Xray photoelectron spectroscopy (XPS), and X-ray powder diffraction (XRD), and the optical and electronic properties of computational models were studied using the time-dependent density functional theory (TD-DFT). The characterization results confirmed the successful doping of nitrogen on the surface of carbon dots. The N-CDs exhibited high affinity toward 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)-diammonium salt (ABTS) with the Michaelis−Menten constant (K M ) of 0.018 mM in a test for their peroxidase-like activity. Particularly, since hydrogen peroxide (H 2 O 2 ) is the oxidative product of cholesterol in the presence of cholesterol oxidase, a sensitive and selective method of cholesterol detection was developed. Overall, the obtained results from TD-DFT confirm the strong adsorption of H 2 O 2 on the graphitic N positions of the N-CDs. The laminated three-dimensional (3D)-μPAD featuring a 6 mm circular detection zone was fabricated using a simple wax screen printing technique. Classification of TC according to the clinically relevant criteria (healthy, <5.2 mM; borderline, 5.2−6.2 mM; and high risk, >6.2 mM) could be determined by the naked eye within 10 min by simple comparison using a color chart. Overall, the proposed colorimetric device serves as a low-cost, rapid, simple, sensitive, and selective alternative for TC detection in whole blood samples that is friendly to unskilled end users.
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