Melatonin levels may be related to the risks of breast cancer and prostate cancer. The measurement of urinary melatonin is also useful in monitoring serum melatonin levels following oral administration. In this work, melatonin is the target molecule, which is imprinted onto poly(ethylene-co-vinyl alcohol) by evaporation of the solvent on the working electrode of an electrochemical sensing chip. This sensing chip is used directly as a tool for optimizing the imprinting polymer composition, flow rate, and injection volume of the samples. Microfluidic sensing of the target and interference molecules revealed that the lowest detection limit is as low as ~pM, and the electrochemical response is weak even at high interference concentrations. Poly(ethylene-co-vinyl alcohol), containing 44 mol. % ethylene, had an imprinting effectiveness of more than six-fold. In random urine analysis, the microfluidic amperometric measurements of melatonin levels with an additional and recovery of melatonin, the melatonin recovery achieved 94.78± 1.9% for melatonin at a concentration of 1.75-2.11 pg/mL.
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