Characterization of peptide release from neuronal cells with microfluidics and mass spectrometry
The nervous system is a complex network of a large number of neurons, and the neuronal communication between these cells often relies on chemical signals. Deciphering these signaling molecules, including neuropeptides, is an important but challenging task. Microfluidic technology allows the manipulation of mass-limited samples and the control of the extracellular microenvironment, making it well suited for studying neurons. In this work, we developed several microfluidic devices that can be interfaced to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) for characterization of neuropeptide release in response to chemical stimulation. One of the microdevices contains an inlet reservoir connected to three sampling channels. Neurons are loaded into the inlet, and the surrounding chemical environment is precisely controlled. The analytes are collected onto a derivatized surface as the media in the reservoir flow through the channel. After that, the surface is interrogated with offline MALDI-MS imaging. This device allows the temporal analysis of the stimulation event, by sequentially sampling the media in different channels. In another device, one single neuron is loaded inside the microchannel, and the releasate is collected in the downstream, enabling single cell analysis. Microfluidics even allows subcellular regions of a neuron to be separately manipulated. In the compartmentalized device, three parallel large channels are connected by small interconnects, and under appropriate conditions, the neurites from cells loaded into the main channel can grow through the interconnects and enter into the adjacent channels. The fluidic environment in each channel can be individually controlled. This dissertation also presents a unique method to quantify peptides based on the adsorption length in a microchannel. The device used here contains a cell chamber and a long serpentine channel. As the ability of a surface to adsorb an analyte is fixed per area, a larger amount of analyte covers a longer section of the channel. Experiments with standards confirmed the expected linear relationship of the sample amount and the adsorption length. By applying this approach to a small number of Aplysia bag cell neurons, the determined amounts of peptides released from a single neuron agreed with other methods. Our quantitation method provides a simple, label-free, and robust way for quantitation of mass-limited samples within microfluidic devices.
Microfluidics, Mass spectrometry, matrix-assisted laser desorption ionization (MALDI), Neuropeptide, Neuron
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign