Analytical Chemistry elegans, the well-studied neural development model, have been placed in microﬂuidic chambers for the interrogation of a number of factors, including odor recognition and Ca2+ imaging,15,16 dynamic cellular processes,17 and high throughput genes and drug screening.18 Finally, several devices have been employed to study the injury and regeneration of neuron processes as well as to model disease within a device.19−21 While microﬂuidic technologies have been used to address a wide range of topics in neuroscience, little of this research has involved the investigation of neuropeptides. A number of detection platforms have been used to investigate neuropeptide release, including enzyme immuno- assays, radioimmunoassays, and capillary electrophoresis coupled to laser-induced ﬂuorescence.22,23 While highly sensitive and quantitative, these techniques require analyte preselection prior to analysis, followed by either a derivatization procedure to render the peptide ﬂuorescent or the creation of an antibody. In contrast, MS provides high sensitivity and information content for the entire peptide complement, without analyte preselection. Matrix-assisted laser/desorption ionization (MALDI) MS oﬀers high salt tolerances and a relative ease of coupling to microﬂuidics. For all of these reasons, MS has proven to be a great detection platform for peptide release studies.24−27 We present a microﬂuidic design that allows the user to selectively apply chemical stimulations to neurons maintained in the device. The resulting neuropeptide releasates are collected oﬀ-line and detected with MALDI- time-of-ﬂight (TOF)-MS. This approach provides information regarding peptide release content and the physiological conditions necessary for release. Previously, we reported several devices that employed MALDI-MS imaging to collect and quantify neuropeptides on chip.28,29 In this study, we achieve further control over the extracellular microenvironment by creating a microvalve-controlled device that exposes a low-density neural network to measured time periods of exposure to a deﬁned chemical stimulation. With the added temporal control over stimulation and collection, the onset of peptide release can be determined and compared. By collecting peptide releasates and characterizing them via MS, oﬀ-chip, multiple-day stimulations can be performed on the same neural network and the speciﬁc released peptides can be characterized. Bag cell neurons from the marine mollusk Aplysia californica are widely used in neuropeptide studies. We exposed these neurons to both general and bag cell-speciﬁc chemical stimulations, followed by the subsequent collection and detection of peptide releasates. We observed both the onset and refractory period of bag cell peptide release for elevated K+ and insulin exposures; our results show a robust diﬀerence in the time frame required to detect peptides after exposure to distinct chemicals. ■ EXPERIMENTAL SECTION Reagents. Chemicals were purchased from Sigma Aldrich (St. Louis, MO), and organic solvents were purchased from Fisher Scientiﬁc (Fairlawn, NJ) unless otherwise noted. The polydimethylsiloxane (PDMS) prepolymer kit, Sylgard 184, was purchased from Dow Corning (Midland, MI). 2- [Methoxy(polyethyleneoxy)propyl]trimethoxysilane (OEG, 90%) was purchased from Gelest, Inc. (Morrisville, PA). Filtered artiﬁcial seawater (ASW, pH 7.8) contained 460 mM NaCl, 10 mM KCl, 10 mM CaCl2, 22 mM MgCl2, 26 mM MgSO4, 2.5 mM NaHCO3, and 15 mM HEPES. Negative photoresist SU-8 2075 and developer were obtained from Article MicroChem Corp. (Newtown, MA). Positive photoresist AZ 4620 and developer were from AZ Electronic Materials Corp. (Somerville, NJ). Device Fabrication. The device consists of two separate layers, a ﬂow channel and a pressure channel layer. Silicon masters were prepared using a traditional photolithographic process as reported previously.30,31 The ﬂow channel master was made with AZ4620 photoresist to a height of 25 μm. Wafers were heated to 200 °C for 20 min to create rounded corners in the channels. Octadecyltrichlorosilane was evapo- rated onto the wafer for 4 h or longer to allow for the removal of PDMS from the wafer. For the pressure channel master, SU- 8 2075 photoresist was spun and patterned to make channels 100 μm in height. Small pieces of silicone tubing (Helix Medical, Carpinteria, CA) were glued onto the SU-8 features on the master. Uncured 20:1 PDMS was spun onto the ﬂow channel master at 2050 rpm for 60 s; 5:1 PDMS was poured onto the pressure channel master. Both masters were allowed to partially cure at 70 °C until just set. Then both PDMS blocks were aligned and adhered together and cured at 90 °C overnight. The main reservoir and outlet were made with an AcuPunch biopsy punch (Acuderm, Inc., Ft. Lauderdale, FL) of 1 mm. The PDMS block was then adhered to a 30 mm cell culture dish (Greiner Bio-One, Monroe, NC), and uncured PDMS was poured around the device to prevent leakage. The device was cured again at 70 °C for 1 h and both the ﬂow and pressure channels were ﬁlled with ASW. Pressure channels were connected to an in-house built pneumatic solenoid valve system that is controlled by LabView software (National Instruments, Austin, TX). The program controls the application or removal of pressure to the pressure channels for closing or opening the channels, respectively. To reduce analyte losses into the PDMS material,29,32 channels were treated with 5:1:1 H2O/HCl/H2O2 for 5 min, rinsed with deionized water (DI; Milli-Q Biocel, Millipore Corporation, Billerica, MA) for 5 min, OEG for 30 min, and ﬁnally ﬂushed with DI water for a minimum of 4 h to yield OEGylated PDMS, as described previously.33 Prior to use, PDMS devices were washed with methanol and dried with N2. Cell Culture Experiments. Aplysia californica (100−150 g; National Resource for Aplysia, Miami, FL) were anesthetized by injecting 390 mM of MgCl2 equal to 1/2 the animal’s body weight. The abdominal ganglia was dissected and incubated at 34 °C in ASW containing 10 mg/mL protease from Streptomyces griseus, 60 mg/L penicillin G, 100 mg/L streptomycin, and 100 mg/L gentamicin for 90 min. This treatment of the ganglia allows for removal of the connective tissue surrounding the bag cell neurons. Individual bag cell neurons were then isolated and loaded into the main reservoir of the microﬂuidic device. A small, thin piece of PDMS was placed over the main reservoir to direct ﬂow through the ﬂow channels. Cells were cultured in ASW for 24 h prior to stimulation experiments. Stimulation Experiments. Two syringes, containing either a solution of 60 mM of KCl in ASW or 5 μM of insulin in ASW, were attached to a syringe pump (Harvard Apparatus, Holliston, MA) and connected to the stimulation channels on the device. A syringe ﬁlled with ASW connected another syringe pump to the inlet reservoir. Pre- and postcontrols were performed with ASW as cell culture media. Each series of chemical stimulation measurements was performed on a network of cultured cells in the microﬂuidic device. Both the ASW and chemical stimulation solution were ﬂowed through 9447 dx.doi.org/10.1021/ac302283u | Anal. Chem. 2012, 84, 9446−9452