In this paper, we successfully applied a cost-effective and simple amperometric sensing design featuring a single microhole-water/organic gel interface to determine native sodium, calcium and potassium ion concentrations in biological fluids. The detection was based on measuring current changes associated with the transfer of each cation type across a microhole supported water/polyvinylchloride-2-nitrophenyl octylether gel interface. Three different sensing platforms were applied to the same biological sample with each only differing in the selective ionophore incorporated into the organic phase: dibenzo-18-crown-6 for sodium, calcium ionophore II (ETH 129) for calcium and valinomycin for potassium ions. Assisted transfer reactions of each ion from the aqueous to the organic gel layer by the corresponding ionophores were first characterized using cyclic voltammetry and then further analyzed using differential pulse stripping voltammetry. A linear dynamic range from 5 mu M to 100 mu M was achieved for each ion target. This enabled quantitative measurements of native concentrations of Na+, Ca2+ and K+ in biological fluid samples including serum and plasma. The amperometric measurements were also validated by comparing with inductively coupled plasma mass spectrometry. (C) 2016 Elsevier B.V. All rights reserved.