In this work, for the first time, a novel electrochemical biosensor was fabricated based on modification of a glassy carbon electrode with multi-walled carbon nanotubes-ionic liquid (MWCNTs-IL) which was used as a platform to the synthesis of molecularly imprinted polymers having glucose (GL), glucose oxidase (GO), cholesterol (CL), cholesterol oxidase (CO), cholesterol esterase (CE), horse radish peroxidase (HRP), and uric acid (UA) as template molecules. For simultaneous biosensing of GL and CL, the biosensor (MIPs/MWCNTs-IL/GCE) was incubated with the enzymes and then, it was individually incubated with GL and CL. Response of the biosensor was based on the reduction of the hydrogen peroxide produced from enzymatic reactions. Individual incubation of the biosensor with GL and CL generated two very similar peaks while its incubation with both GL and CL generated a single peak which needed to be assisted by first- and second-order calibration methods to choose the best method. Second-order hydrodynamic differential pulse voltammetric data were generated and modeled by MCR-ALS, PARASIAS, and PARAFAC2 to simultaneous determination of them in the presence of UA as uncalibrated interference based on exploiting second-order advantage. Amperometric responses of the biosensor to GL and CL were also calibrated and assisted by first-order calibration methods for simultaneous determination of GL and CL in the presence of UA based on exploiting first-order advantage. The result confirmed that the biosensor assisted by second-order data modeled by MCR-ALS showed the best performance for simultaneous determination of CL and GL in the presence of UA as uncalibrated interference in both synthetic samples and human serum samples which was comparable with HPLC-UV as the reference method. It should be noted the biosensor was able to simultaneous determination of CL and GL in the ranges of 0.5–15 pM and 1–18 pM with limits of detection of 0.81 pM and 0.23 pM, respectively, and response of the biosensor was stable, selective, ultrasensitive, repeatable, and reproducible. This work as the first report chemometric assisted enzymatic biosensing of GL and CL can be continued to develop more projects for biomedical purposes.
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