Electrochemical biosensors for on-chip detection of oxidative stress from immune cells

Abstract

Seamless integration of biological components with electrochemical sensors is critical in the development of microdevices for cell analysis. The present paper describes the integration miniature Au electrodes next to immune cells (macrophages) in order to detect cell-secreted hydrogen peroxide (H(2)O(2)). Photopatterning of poly(ethylene glycol) (PEG) hydrogels was used to both immobilize horseradish peroxidase molecules onto electrodes and to define regions for cell attachment in the vicinity of sensing electrodes. Electrodes micropatterned in such a manner were enclosed inside poly(dimethylsiloxane) fluid conduits and incubated with macrophages. The cells attached onto the exposed glass regions in the vicinity of the electrodes and nowhere else on the non-fouling PEG hydrogel surface. A microfluidic device was converted into an electrochemical cell by placing flow-through Ag∕AgCl reference and Pt wire counter electrodes at the outlet and inlet, respectively. This microdevice with integrated H(2)O(2)-sensing electrodes had sensitivity of 27 μA∕cm(2) mM with a limit of detection of 2 μM. Importantly, this microdevice allowed controllable seeding of macrophages next to electrodes, activation of these cells and on-chip monitoring of H(2)O(2) release in real time. In the future, this biosensor platform may be utilized for monitoring of macrophage responses to pathogens or for the study of inflammatory signaling in micropatterned cell cultures.

Figure 1

(a) Upper panel: schematic of a single electrode for detection of cell secreted H₂O₂. Macrophages are captured on a micropatterned surface in the vicinity of sensing electrode. The sensor consists of an HRP-carrying hydrogel disc immobilized on top of 300 μm diameter Au electrode. Each channel constituted an electrochemical cell with Ag/AgCl flow-through reference and Pt wire counter electrodes placed at the outlet and inlet, respectively. Lower panel: There were two parallel microfluidic channels in the device, with 4 individually addressable electrodes in each microfluidic channel. Each fluidic channel had a volume of 3 μl. (B) Step-by-step diagram for immobilizing HRP carrying hydrogel microstructures on top of Au electrodes.

Figure 2

(a and b) Au electrodes (300 μm diameter) integrated with hydrogel microstructures (600 μm diameter). A 300 μm wide ring around each electrode served as a site for cell attachment. (c) Brightfield/fluorescence overlay image showing fluorescently labeled protein (avidin-Alexa546 (red)) deposited in the site of cell attachment. This picture highlights that hydrogel structures were non-fouling and that proteins adsorbed only on the exposed glass regions. This micropatterning strategy was used to reproducibly position cells next to electrodes.

Figure 3

(a) The effect of HRP loading on the biosensor response. Ratios represent v/v of HRP in PEG prepolymer solutions. Hydrogel-coated Au electrodes were poised at −0.2V vs. Ag/AgCl references and were challenged with 200 μM H₂O₂. Amperometric responses from different electrode formulations were normalized by the response from a 1:10 HRP:PEG hydrogel coating. (b) Electrical responses of electrodes to 200 μM H₂O₂ at working potentials ranging from −0.3 to 0.6 V vs. Ag/AgCl reference. Electrodes coated with HRP-PEG were compared to electrodes covered with PEG hydrogel only.

Figure 4

(a and b) Amperometric responses of HRP/hydrogel/Au electrodes to 5 μM and 100 μM H₂O₂. (b) Calibration curve of current vs. analyte concentration. HRP/hydrogel/Au electrodes showed sensitivity of 27 μA/mM·cm² and the detection limit of ∼2 μM H₂O₂. In both sets of experiments electrodes were poised at −0.2 V vs. Ag/AgCl.

Figure 5

(a) Macrophages attaching next to HRP-containing hydrogel/Au electrode. Cell nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue color). (b) A smaller magnification image showing two electrodes integrated with cells. (c) Amperometric responses obtained from stimulated macrophages cultured next to sensing miniature electrodes. Measurements of electrode response to macrophages without stimulation and media with stimulant (PMA) but no cells were carried out in parallel, in the same device as cell detection experiment, and were used as controls. As seen from these negative current due to reduction of H₂O₂ was only observed in the presence of activated macrophages. Electrodes were poised at −0.2 V vs. Ag/AgCl.