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

The production of reactive oxygen species (ROS) in the body has been shown to play a significant role in the development and progression of numerous diseases. This makes it important to develop a method of detection for hydrogen peroxide (H2O2), the most stable ROS. Several methods such as the use of fluorescent probes and electrochemistry have been utilized in the past to detect the imbalance in ROS levels generated from injured or stimulated cells. An imbalance in the levels of ROS leads to a state of oxidative stress within the body. Different enzymes such as horseradish peroxidase (HRP) and superoxide dismutase have been used in the detection of ROS. HRP is commonly used as the biorecognition element in many H2O2 sensors. Researchers have looked into immobilizing these enzymes onto carbon nanotubes and nanoparticles to increase sensor sensitivity. In this chapter, we present experimental procedures to perform electrochemical quantification of H2O2, one of the major ROS release from injured cells (macrophages and hepatocytes).

Miniature enzyme-based electrodes for detection of hydrogen peroxide release from alcohol-injured hepatocytes.

Alcohol insult to the liver sets off a complex sequence of inflammatory and fibrogenic responses. There is increasing evidence that hepatocytes play a key role in triggering these responses by producing inflammatory signals such as cytokines and reactive oxygen species (ROS). In the present study, we employed a cell culture/biosensor platform consisting of electrode arrays integrated with microfluidics to monitor extracellular H(2)O(2), one of the major ROS types, produced by primary rat hepatocytes during alcohol injury. The biosensor consisted of hydrogel microstructures with entrapped horseradish peroxidase (HRP) immobilized on an array of miniature gold electrodes. These arrays of sensing electrodes were integrated into microfluidic devices and modified with collagen (I) to promote hepatocyte adhesion. Once seeded into the microfluidic devices, hepatocytes were exposed to 100 mM ethanol and the signal at the working electrode was monitored by cyclic voltammetry (CV) over the course of 4 h. The CV experiments revealed that hepatocytes secreted up to 1.16 µM H(2)O(2) after 3 h of stimulation. Importantly, when hepatocytes were incubated with antioxidants or alcohol dehydrogenase inhibitor prior to alcohol exposure, the H(2)O(2) signal was decreased by ~5-fold. These experiments further confirmed that the biosensor was indeed monitoring oxidative stress generated by the hepatocytes and also pointed to one future use of this technology for screening hepatoprotective effects of antioxidants.

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

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.