A microfluidic method for dopamine uptake measurements in dopaminergic neurons

Abstract

Dopamine (DA) is a classical neurotransmitter and dysfunction in its synaptic handling underlies many neurological disorders, including addiction, depression, and neurodegeneration. A key to understanding DA dysfunction is the accurate measurement of dopamine uptake by dopaminergic neurons. Current methods that allow for the analysis of dopamine uptake rely on standard multiwell-plate based ELISA, or on carbon-fibre microelectrodes used in in vivo recording techniques. The former suffers from challenges associated with automation and analyte degradation, while the latter has low throughput and is not ideal for laboratory screening. In response to these challenges, we introduce a digital microfluidic platform to evaluate dopamine homeostasis in in vitro neuron culture. The method features voltammetric dopamine sensors with limit of detection of 30 nM integrated with cell culture sites for multi-day neuron culture and differentiation. We demonstrate the utility of the new technique for DA uptake assays featuring in-line culture and analysis, with a determination of uptake of approximately ∼32 fmol in 10 min per virtual microwell (each containing ∼200 differentiated SH-SY5Y cells). We propose that future generations of this technique will be useful for drug discovery for neurodegenerative disease as well as for a wide range of applications that would benefit from integrated cell culture and electroanalysis.

A microfluidic platform for continuous monitoring of dopamine homeostasis in dopaminergic cells

“…Numerous microchannel-based microfluidic platforms have been used to evaluate neurotransmitter release from neuronal cells 20,21 , but these platforms have not been applied to this problem; moreover, we are unaware of any system that is capable of evaluating both neurotransmitter uptake and release. The closest system was our previous method 19 , which had separate modules for (a) cell culture and (b) DA electroanalysis. Here, we sought to combine (a) and (b) into a single, integrated unit known as a “cell-culture/electroanalytical sensor” (or “c-e-sensor”) that allows for the continuous monitoring of DA homeostasis.…”

“…In response to the challenge described above, we developed an automated digital microfluidic (DMF) platform that integrates in vitro cell culture 15,16 with electrochemical analysis 17,18 . We recently described an initial solution to this problem 19 , i.e., a microfluidic device that includes (1) a cell-culture module designed for multi-day dopaminergic neuron culture and (2) a quantitative amperometric DA sensor (“e-sensor”) module. A key limitation to this previous method 19 is its modular nature, i.e., droplets of culture medium are shuttled between the two modules, rendering continuous measurements impossible.…”

“…We recently described an initial solution to this problem 19 , i.e., a microfluidic device that includes (1) a cell-culture module designed for multi-day dopaminergic neuron culture and (2) a quantitative amperometric DA sensor (“e-sensor”) module. A key limitation to this previous method 19 is its modular nature, i.e., droplets of culture medium are shuttled between the two modules, rendering continuous measurements impossible. Here, we introduce a substantially improved system that allows for multi-day cell culture and differentiation with real-time continuous measurements of DA uptake.…”

PortaDrop: A portable digital microfluidic platform providing versatile opportunities for Lab-On-A-Chip applications

“…To our knowledge, this is the first time EIS was used to monitor the media exchange during passive dispensing in a virtual microwell. Passive dispensing is a well-known mechanism in DMF to allow precise fluid incubation underneath a hydrophilic spot in the top chip [ 14 , 53 , 55 , 57 , 71 , 72 ]. Barbulovic-Nad et al showed by fluorescence measurements that three source droplets are necessary to fully exchange the volume of the virtual microwell completely [ 14 ].…”

“…They integrate an Ag/AgCl on-chip pseudo-reference electrode into the DMF device and use voltammetry achieving a sensor response close to Nernstian behavior. Yu et al monitor the dopamine uptake in neurons by cyclic voltammetry (CV) integrated in DMF [ 53 , 54 ]. Dryden et al use integrated electrodes for linear sweep voltammetry (LSV) measurements of different acetaminophen concentrations generated by repeated dilution using the EWOD fluid handling capabilities bearing a limit of detection of 76 μM [ 55 ].…”