Real-time monitoring of neuronal cells through electrohydrodynamic patterning of flexible graphene microelectrodes
Investigation of the change in the electrochemical properties of neuronal cells upon exposure to stress factors imparts vital information about the stages prior to their death. This study presents a graphene-based biosensor for real-time monitoring of N27 rat dopaminergic cells which characterizes cell adhesion and cytotoxicity factors through impedance spectroscopy. The aim was to monitor the growth of the entire cell network via a non-metallic flexible electrode with high temporal resolution. Therefore, a water-based graphene solution was formulized as a conductive ink, 3D printed into a flexible substrate through a novel electrohydrodynamic approach, resulting in electrodes with a conductivity of 6750 s/m. A scalable and aqueous phase exfoliation of graphite to high yield and quality of few layer graphene (FLG) was achieved through Bovine Serum Albomine (BSA) and wet ball milling. The produced graphene ink is tailored for printable and flexible electronics, having shown promising results in terms of electrical conductivity and temporal stability. Shear force generated by steel balls resulted in 2-3 layer defect-free graphene platelets with an average size of hundreds of nm and with concentration of about 5.1 mg/ml characterized by Raman spectroscopy, atomic force microscopy (AFM), transmittance electron microscopy (TEM) and UV-vis spectroscopy. Further, a conductive ink was prepared and printed on flexible substrate (Polyimide) with controlled resolution. Scanning electron microscopy (SEM) and Profilometry revealed the effect of thermal annealing on the prints to concede consistent morphological characteristics. The resulted sheet resistance was measured to be Rs =36.75 Ω/sqr for prints as long as 100 mm. Printable inks were produced in volumes ranging from 20 ml to 1 L with potential to facilitate large scale production of graphene for applications in biosensors as well as flexible and printable electronics.
The presented high-throughput method enabled micro-scale monitoring of the entire cell network via the design of a PDMS-based growth channels. The electrical resistance of the cell network was measured continually along with their network density, constituting a mean density of 1890 cell per square mm at full cell confluency. The results demonstrate the applicability of the impedance-based sensing of the cell network for rapid screening of the cytotoxic elements, and the real-time effect of UV exposure on dopaminergic neurons was reported as an immediate application of the device. Most notably, we were interested in creating a layered microstructure through inkjet printing the water-dispersed graphene to enhance the sensitivity to damage detection in neuronal cells. The microfluidic deposition of graphene electrodes could provide texture cues that are favored by the cells while the patterns are consolidated through the electrostatic field to maintain the stability of the microelectrodes upon flexure. Thus, the detection of detachment can be studied with higher precision through impedance spectroscopy technique, where the cell body is treated as a physical particle which impedes the electrical current passing through the electrode array. Accordingly, inflicting cellular damage leads to alteration in membrane morphology, cellular shrinkage, detachment, and lift-off which will affect the cellular impedance. This cellular damage could be detected by the presented methodology with temporal resolution of a minute.