A printed superoxide dismutase coated electrode for the study of macrophage oxidative burst

Abstract

The miniaturization of electrochemical sensors allows for the minimally invasive and cost effective examination of cellular responses at a high efficacy rate. In this work, an ink-jet printed superoxide dismutase electrode was designed, characterized, and utilized as a novel microfluidic device to examine the metabolic response of a 2D layer of macrophage cells. Since superoxide production is one of the first indicators of oxidative burst, macrophage cells were exposed within the microfluidic device to phorbol myristate acetate (PMA), a known promoter of oxidative burst, and the production of superoxide was measured. A 46 ± 19% increase in current was measured over a 30 min time period demonstrating successful detection of sustained macrophage oxidative burst, which corresponds to an increase in the superoxide production rate by 9 ± 3 attomoles/cell/s. Linear sweep voltammetry was utilized to show the selectivity of this sensor for superoxide over hydrogen peroxide. This novel controllable microfluidic system can be used to study the impact of multiple effectors from a large number of bacteria or other invaders along a 2D layer of macrophages, providing an in vitro platform for improved electrochemical studies of metabolic responses.

Figure 1

The effect of enzymatic activity on oxidative current when exposed to H₂O₂ or O₂⁻. All LSVs were performed with SPE in PBS at a scan rate of 20 mV/s from 0.2 to 0.7 V. The PBS background was subtracted to yield net current change which is shown here. The net response of an unmodified Pt electrode to O₂⁻ (dashed line, O₂⁻ produced with 1 µM XOD and 100 µM HPX) is compared with the response of a SOD electrode to O₂⁻ (black line, O₂⁻ produced with 1 µM XOD and 100 µM HPX). These electrodes were compared with the response of the SOD electrode in H₂O₂ (grey line, 1 µM H₂O₂). A net difference of 0.93 µA at 0.49 V was seen between the response of the SOD electrode in O₂⁻ and H₂O₂.

Figure 2

Amperometric calibration of hand-cast and IJP SOD biosensors. The handcast film (A) demonstrates greater deviation in slope (3.1 ± 0.9 nA/µM HPX, 28% RSD) compared with (B) electrodes coated with a printed film (3.5 ± 0.4 nA/µM HPX, 11% RSD). The inset (A) is a schematic showing the SPE, with each SOD coated electrode designated with a square, circle or triangle, used in the calibration curves. The counter electrode and an unused 0.08 mm² band electrode are also shown.

Figure 3

Macrophage response to PMA exposure in a PDMS microfluidic device. Two chambers were run concurrently where one chamber had no cells (black) and the other had 5×10⁵ macrophages (grey). (A) The raw amperometric response of macrophages over two hours. (B) The absolute change in current during stop-flow, where the baseline current is subtracted from the peak current. The change in current (B) corresponds to an increase of O₂⁻ production by 9 ± 3 attomoles/cell/sec.