Flexible Multimodal Neural Probe with Integrated Three-Electrode Aptameric Sensing for In Vivo Monitoring of Dopamine Dynamics and Neural Activity.

Neural function emerges from the interplay between electrical activity and neurochemical signaling, yet most implantable neural interfaces primarily record electrophysiological signals and lack molecular specificity for neurotransmitter monitoring. Here, we developed a flexible multimodal neural probe integrating an on-chip three-electrode electrochemical aptamer sensor with electrophysiological recording sites for combined monitoring of extracellular dopamine (DA) dynamics and neuronal activity. Fabrication of the electrochemical interface was systematically optimized by controlling gold nanostructure (AuNS) electrodeposition from 0.50 to 0.70 V. Electrodeposition at 0.65 V provided the best balance between increased electrochemically active surface area (ECSA), surface morphology, fabrication reproducibility, and electrical isolation, yielding an approximately 4.7-fold ECSA enhancement with a probe-to-probe coefficient of variation (CV%) of 2.02%. A methylene blue-labeled DA aptamer was assembled onto the AuNS working electrodes for sequence-specific molecular recognition, while integrated Ag/AgCl reference and nanostructured platinum counter electrodes completed the on-chip sensing system. Electrochemical impedance analysis further demonstrated reproducible interfacial characteristics following stepwise functionalization. The electrophysiological electrodes exhibited a mean impedance of 366.02 ± 18.63 kΩ at 1 kHz with an interprobe CV% of 5.09%. Square-wave voltammetry frequency was experimentally optimized from 10 to 200 Hz, with 100 Hz providing the best balance between analytical response, background current, and reproducibility. Under optimized conditions, the sensor demonstrated sequence-specific DA recognition, selectivity against electroactive interferents, and an experimentally determined detection limit of 10 fM. Two absolute linear response regions were identified at 0.5-10 pM (R2 = 0.9945) and 0.5-10 nM (R2 = 0.9949), while the broader detectable concentration range extended from 10 fM to 1 μM. Flow-injection and brain-phantom experiments demonstrated reversible DA sensing. In acute rat caudate-putamen experiments, intravenous nomifensine induced DA-associated electrochemical responses accompanied by increased neuronal firing and γ/high-γ local field potential activity, supporting this platform for acute multimodal neurochemical and electrophysiological interrogation in vivo.