Potential-pulse-assisted co-immobilization of multiple aptamers on microelectrode arrays for multiplexed neurotransmitter detection

Accurate and simultaneous determination of multiple neurotransmitters (NTs) is crucial for a thorough understanding of brain functions and for diagnosing neurological disorders. We have developed an electrochemical biosensor utilizing aptamer recognition for the highly sensitive and simultaneous detection of multiple neurotransmitters, including glutamate quantification for the first time in multiplex detection. Microelectrode arrays (MEAs) served as biosensing platforms to facilitate the recording of signals from multiple channels, rapid mass transfer rates, and high spatial resolution, fundamental for studying NTs release in nervous tissue. To enhance aptamer receptor loading, gold nanostructures were electrodeposited onto the microelectrodes, enhancing the active surface area and electrode morphology. A potential-pulse-assisted method was employed to achieve site-selective immobilization of three different aptamers on a single MEA chip within 30 min, enabling fast and reproducible sensor fabrication. This unique strategy ensured aptamer-specific immobilization, minimized cross-talk, and allowed for multiplex detection of serotonin, glutamate, and dopamine with high sensitivity and specificity. Additionally, using polyethylene glycol (PEG) as a blocking molecule, the aptamer-functionalized MEAs showed enhanced antifouling properties and maintained detection capabilities in complex environments. This multiplexed detection strategy enables a high-performance and robust biosensor platform with strong clinical relevance, offering substantial potential for in vivo monitoring of neurotransmitter release and the diagnosis of neurological disorders.