MP39:Quantitation and Spatial Control of Peptide Release

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Ming Zhong and Jonathan V. Sweedler
Department of Chemistry and Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL

Within the brain, communication between neuronal cells relies on a variety of signaling molecules, one important category of which is neuropeptides. Different regions of an individual neuron experience distinct chemical environments and the overall cellular response depends on these chemical microenvironments. Measuring these peptides of low abundance and providing chemical and spatial control of these small-volume regions around cultured neurons remain challenges. Microfluidics is a suitable solution because of the minimal sample consumption and the ability to manipulate microenvironments. First, we demonstrate a microfluidic device capable of quantifying peptide release by measuring the length of channel covered by each peptide. In this device, neurons are placed in small chambers, and the cellular releasates are collected as they flow along a serpentine channel that contains a surface derivatized with octadecyltrichlorosilane designed to collect the peptides released from chemically-stimulated neurons. In order to minimize peptide loss due to nonspecific adsorption, the sample chamber surface is patterned with oligo(ethylene glycol). Next the derivatized surfaces are characterized by matrix-assisted laser desorption/ionization (MALDI)-based mass spectrometry (MS) imaging to determine the part of channel covered by a peptide. By carefully controlling the flow rate, experiments with known amounts of acidic peptide (AP) show a linear correlation between the length of channel occupied by AP and its amount. We then use our approach with neurons from Aplysia californica, a model organism for neurochemistry research. For one example using a small number of Aplysia bag cell neurons stimulated by elevated potassium, the expected signaling peptides, including AP and alpha-bag cell peptide (BCP) are detected. The amounts of released peptides determined by the channel coverage length match other measures of peptide release amounts. Therefore, this microfluidic device offers a unique label-free solution to quantifying mass-limited samples. In order to selectively manipulate the environments surrounding sub-regions of a single neuron, we are adapting a new microfluidic platform, which has the cells loaded and cultured inside a main channel and contains two other parallel channels in which the liquid flows can be separately manipulated. Those secondary channels are interconnected to the main channel using a large number of small-diameter interconnections that have controlled surface chemistry and can selectively guide the axons and dendrites into these secondary channels. Fluidic isolation of a channel is achieved by creating a minute hydrostatic pressure difference from the adjacent channels. This type of microfluidic device allows controlled delivery of reagents to isolated subcellular regions and collection of peptide releasate locally.

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