Simultaneous detection of cell-secreted TNF-α and IFN-γ using micropatterned aptamer-modified electrodes

Cellular production of such cytokines as interferon (IFN)-γ and tumor necrosis factor (TNF)-α is used to determine disease-specific immune responses and may be used to diagnose infectious diseases such as tuberculosis. In this paper, we describe the development of micropatterned electrodes functionalized with electroactive aptamers for multiplexed detection of immune-cell-produced cytokines. A sequence of electrode deprotection and aptamer incubation steps were used to assemble anti–IFN–γ DNA aptamers and anti-TNF-α RNA aptamers on individually addressable half-ring electrodes. Aptamer molecules were thiolated for assembly on gold and were functionalized with methylene blue redox reporter for electrochemical signal transduction. Specificity of individual sensors to the correct cytokine species was confirmed by exposure to recombinant cytokines. For cell detection experiments, electrode arrays were integrated into microfluidic devices and incubated with immune cells. Design of the surface was such that a small group of ∼400 cells attached in the circular adhesion sites surrounded by half-ring electrodes sensing IFN-γ and TNF-α. The microdevice consisted of two parallel microfluidic channels, each channel containing four cell capture/sensing sites. Upon mitogenic activation, secreted IFN-γ and TNF-α molecules were monitored by performing square wave voltammetry (SWV) at different time points at individually addressable electrodes. This biosensing platform was used to analyze the quantity and rate of cytokine release from primary T cells and a monocyte cell line. Upon further development of this platform may be enhanced to enable detection of larger number of cytokines and used to correlate the levels and dynamics of cytokine release in immune cells to diagnosis and treatment of infectious diseases.