Article pubs.acs.org/ac Microﬂuidic Device for the Selective Chemical Stimulation of Neurons and Characterization of Peptide Release with Mass Spectrometry Callie A. Croushore,† Sam-ang Supharoek,†,‡ and Jonathan V. Sweedler*,† Chang Young Lee, †,§ Jaroon Jakmunee, ‡ †Department of Chemistry and Beckman Institute, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States ‡Department of Chemistry, and Center of Excellence for Innovation in Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand §School of Nano-Bioscience and Chemical Engineering, Interdisciplinary School of Green Energy, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798, Republic of Korea ABSTRACT: Neuropeptides are synthesized in and released from neurons and are involved in a wide range of physiological processes, including temperature homeostasis, learning, memory, and disease. When working with sparse neuronal networks, the ability to collect and characterize small sample volumes is important as neurons often release only a small proportion of their mass-limited content. Microﬂuidic systems are well suited for the study of neuropeptides. They oﬀer the ability to control and manipulate the extracellular environment and small sample volumes, thereby reducing the dilution of peptides following release. We present an approach for the culture and stimulation of a neuronal network within a microﬂuidic device, subsequent collection of the released peptides, and their detection via mass spectrometry. The system employs microvalve-controlled stimulation channels to selectively stimulate a low-density neuronal culture, allowing us to determine the temporal onset of peptide release. Released peptides from the well-characterized, peptidergic bag cell neurons of Aplysia californica were collected and their temporal pattern of release was characterized with matrix-assisted laser desorption/ ionization time-of-ﬂight mass spectrometry. We show a robust diﬀerence in the timing of release for chemical solutions containing elevated K+ (7 ± 3 min), when compared to insulin (19 ± 7 min) (p < 0.000 01). N molecules that are involved in cell-to-cell communication europeptides are an important class of signaling in both the central and peripheral nervous systems. They are synthesized and stored in neurons and, upon stimulation, are exocytotically released from the cells to function as neuro- transmitters, neuromodulators, trophic factors, and neuro- hormones. Known to inﬂuence a wide range of physiological processes, neuropeptides inﬂuence learning and memory, food intake, pain, and disease.1−3 Identifying neuropeptides that both establish and enrich neuron−neuron interactions and understanding the physical and chemical conditions necessary for their release will aid in uncovering the key eﬀectors of formation and repair in neural networks. Characterizing neuropeptide release in vitro from low-density culturing environments allows cells of interest to be isolated from their potentially heterogeneous neighbors, which oftentimes contain a diﬀerent chemical complement from nearby cells.4 The study of neuropeptide signaling can be challenging; because only a fraction of the mass-limited neuropeptide content of a cell is released after stimulation, sample amounts can be low. Furthermore, in the commonly employed dish- based culturing systems, released peptides are diluted by orders of magnitude before collection, rendering their characterization diﬃcult. There is a need for neuronal culture, collection, and detection systems that both identify released neuropeptides and provide information regarding release conditions. Here we describe an approach to maintain a neuronal network in a controlled environment, selectively apply chemical stimulations, collect peptide releasate with minimal dilution, and couple the releasate collection to oﬀ-line mass spectrometry (MS) for peptide characterization. Microﬂuidic devices are well suited for examining neuro- peptide release. With the evolution of microvalves, ﬂuid control, surface patterning, and chemical gradients, these devices provide the ability to precisely control the micro- environments around neurons while signiﬁcantly reducing sample volumes.5−8 By enabling detection of these mass- limited samples, we gain a broader understanding of peptide release conditions. Additionally, these devices oﬀer a number of advantages that improve cell culture: biocompatibility, gas permeability, and optical transparency. In recent years, progress has been made toward integrating microﬂuidics and neuroscience with cell culture, manipulation, and electrophysiology studies.9−13 Compartmentalized devices ﬂuidically isolate cell bodies from processes, allowing these structures to be examined separately.14 Whole Caenorhabditis Received: August 8, 2012 Accepted: September 24, 2012 Published: September 24, 2012 © 2012 American Chemical Society 9446 dx.doi.org/10.1021/ac302283u | Anal. Chem. 2012, 84, 9446−9452