Electrochemical Biosensor-Based Evaluation of Cholinergic Biomarkers in Experimental Nephropathy

This study presents a rapid method for evaluating cholinesterase (ChE) activity using an amperometric choline biosensor based on an enzymatic membrane incorporating choline oxidase (ChOx) and a selectivity-providing acetylated cellulose layer. The developed biosensor shows a linear response range of 5-500 μM for choline with a sensitivity of 32.2 ± 0.3 μA/(mM cm2), enabling the determination of choline in serum. The biosensor was also adapted for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) determination, achieving LODs of 0.003 and 0.005 U/ml, respectively, with a linear range up to 0.5 U/ml. Notably, the biosensor retains 88% of its initial sensitivity after 28 days of use. Two experimental rat nephropathy models were investigated: mild nephropathy induced by folic acid and advanced nephropathy induced by doxorubicin. A strong correlation between the proposed biosensor and colorimetric analysis was observed in rat serum samples (R2 = 0.988 for ChE; R2 = 0.979 for BChE), confirming its reliability and suitability. Biochemical parameter analysis of rat serum samples revealed a positive correlation between total protein levels and ChE/BChE activity, and a negative correlation between C-reactive protein, ALT, and TBK-AP levels and ChE/BChE activity. Furthermore, ChE and BChE activity was closely associated with nephropathy severity markers, including serum creatinine and urea concentrations. Increasing nephropathy severity was associated with a progressive decrease in cholinesterase activity. These findings highlight ChE activity as a potential indicator of nephropathy progression as well as hepatotoxicity and demonstrate the applicability of the proposed choline biosensors as accurate and efficient tools for wide-profile point-of-care biochemical analysis.