A highly sensitive electrically driven electrochemiluminescent assay for quantification of bile acids in human serum

Abstract

A capillary electrically driven assay with electrochemiluminescent (ECL) detection for total bile acids in human serum was developed and fully validated. Quantification was performed by multiple reactions. First, the bile acids react with nicotinamide adenine dinucleotide (NAD⁺) under catalysis of 3α-hydroxysteroid dehydrogenase (3α-HSD), which is converted to 3-ketosteroid and concomitantly NAD⁺ turns into reduced nicotinamide adenine dinucleotide (NADH). And then Ru(bpy)₃²⁺ is oxidized to be Ru(bpy)₃³⁺, which serves as an electron mediator, and reacts immediately with NADH coexisting in a carrier solution and is converted to Ru(bpy)₃^2+*. NADH transfers an electron to form NAD⁺ and the unstable excited-state species, Ru(bpy)₃^2+*, which emits photons and gives out light when it decays to the ground state, Ru(bpy)₃²⁺. Consequently, the concentration of total bile acids could be determined by the electrochemiluminescent intensity. The assay was linear from 0.1 fmol L⁻¹ to 1000 fmol L⁻¹, with a detection limit of 0.02 fmol L⁻¹. The intra-day and inter-day precision had a coefficient of variation of less than 5.0%. The developed ECL assay had an acceptable correlation with an enzymatic cycling method commonly adopted in clinics for the determination of total bile acids (r = 0.7216). Based on the above-mentioned principle, we established a simple, accurate and highly sensitive approach for the determination of total bile acids. Furthermore, this assay has been applied successfully to the detection of total bile acids in human serum, indicating its practicality for bioanalysis.