Micropatterned Aptasensors for Continuous Monitoring of Cytokine Release from Human Leukocytes

We report the development of a microdevice for detecting local interferon gamma (IFN-gamma) release from primary human leukocytes in real time. Our microdevice makes use of miniature aptamer-modified electrodes integrated with microfluidics to monitor cellular production of IFN-gamma. The aptamer species consists of a DNA hairpin molecule with thiol groups on the 3'-end for self-assembly onto Au electrodes. A redox reporter is covalently attached at the 5'-end for electrochemical sensing. This aptasensor has excellent sensitivity for IFN-gamma (<60 pM detection limit) and responds to the target analyte in real time without additional washing or labeling steps. Aptamer-functionalized electrode arrays are fabricated on glass slides containing poly(ethylene glycol) (PEG) hydrogel patterns designed to expose glass regions adjacent to electrodes while protecting the remainder of the surface from nonspecific adsorption. The micropatterned substrates are integrated with PDMS microfluidic channels and incubated with T-cell-specific antibodies (Ab) (anti-CD4). Upon injection of blood, leukocytes are bound to Ab-modified glass regions in proximity to aptasensors. Cytokine release from captured cells is triggered by mitogenic activation and detected at the aptamer-modified electrodes using square wave voltammetry (SWV). The IFN-gamma signal is monitored in real time with signal appearing as early as 15 min poststimulation from as few as 90 T cells. The observed IFN-gamma release profiles are used to calculate an initial IFN-gamma production rate of 0.0079 pg cell(-1) h(-1) upon activation. The work described here represents an important step toward development of aptasensors for immune cell analysis and blood-based diagnostics.