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Bendable Bioplatform for Electrically stimulated Neuronal Differentiation

Periodic Reporting for period 1 - BEND (Bendable Bioplatform for Electrically stimulated Neuronal Differentiation)

Reporting period: 2016-08-15 to 2018-08-14

Self-health monitoring and Point of care diagnosis (PoC) are focus areas for wearable/implantable devices as they allow monitoring of analytes from closer vicinity of a patient. However, the state-of-the-art PoC devices employ chemically or mechanically thinned compatible metal wafer or synthetic polymer which lacks compatibility enough to be conformable and even develop allergic or inflammatory responses to the patient. So, compatibility of state-of-the-art implantable/wearable devices remains as major challenge in self-health monitoring comfortably. The use of biopolymer-based electronics platform could be a major focus in solving this critical issue.
This Marie Curie Individual fellowship (Project ID: BEND 704807) proposed a bendable bioplatform for holding the electronic circuitry for the development of wearable/implantable electronic devices. Herein, a chitosan biopolymer based biocompatible platform with tunable degradability was developed as bioresorbable patch. The chitosan suspension was drop-casted on a carrier wafer and allowed overnight for air drying to yield chitosan substrate. Then the substrate was chemically cross-linked with Glutaraldehyde for attaining stability in aqueous medium. Biocompatibility of the fabricated platform was evaluated with in vitro human dermal cell adhesion and proliferation. Interdigitated Au electrode established Chi substrate was employed for electrically stimulated neurite differentiation. The Chi based electrode was also employed for cell health monitoring. With these results, we demonstrated that the developed bioplatform could be useful for holding the wearable/implantable devices without any compatibility issues.
In 2016-2018, chitosan (Chi) based bendable bioplatform was fabricated for cell adhesion, spreading, proliferations and electrical stimulation. In particular, during the first year of the fellowship, a bioresorbable, biodegradable, biocompatible and readily conformable Chi substrate was achieved. Major activities in first year of the fellowship were focused on WP-1 and WP-2 for achieving Deliverables D1 to Deliverables 6.
Chi powder was solubilized in acidic aqueous solution (1% acetic acid) and casted on petri dishes and allowed for air drying overnight to yield thin, clear, conformable substrate with smooth surface topography. Aqueous solubility was overcome with crosslinking process while stability in acetone utilized in dissolving sacrificial layer during metallization process. Compatibility of the substrate was assessed with HDF cell adhesion and proliferation. The ultra-thin Chi substrate was fabricated on silicon wafer was used for metallization to realize Au micro-gap electrodes on it.
In vitro biocompatibility assay showed poor adhesion, spreading and proliferation of cell on glutaraldehyde cross-linked scaffold. Whereas, farm adhesion, well spreading and enhance proliferation was noticed when glutaraldehyde cross-linked substrate were blocked in 1M Gly. Thus, the interdigitated platform was functionalized with adhesion molecule (RGD peptide) which results farm cell adhesion and enhanced spreading and proliferation.
In the second year of this fellowship, we realized electrophysiological state of living cell and thereby explored the possibilities of regulating neuronal differentiation mechanism using electrical stimulation from external sources. For this, conductivity of the membrane was achieved with establishing a nanoscale conductive Au layer (15nm) on it and thus the Chi substrate was prepared for electrical stimulation study.
Major activities in second year of the fellowship were focused on WP-2 partly, WP-3 and WP-4 for achieving Deliverables D7 to Deliverables 10:
The cell immobilized Chi substrate was subjected to electrochemical investigation using CV. The CV obtained from cell cultured Chi-GO substrate showed a quasi-reversible redox peak with cathodic peak (Ipc) at +300mV and anodic peak (Ipa) at -300mV. The absence of such peak from a device without cell confirms that redox is originated from the immobilized HDF. The redox peak showed stability to scan rates and scan cycle. This indicates that the electrical signal is stable and repeatable. Considering stability and repeatability redox peak, together with mechanical properties discussed earlier, the device is suitable for in vivo application.
This device is able to monitor cyto-physiologic state by analyzing and quantifying the redox signal. Besides, the microscopic imaging technique was employed for the morphologic investigation of neurite differentiation. Images obtained with phase contrast microscopy showed neurites structure of a differentiated live neuron whereas the scanning microscopic images from fixed cell showed the more details neurite differentiation. The molecular characterization of nerite specific markers has not done yet because of time limitation of the project. I am still continuing the work in my home institute for the molecular confirmations of the electrically stimulated neurite differentiation.
Apart from these research activities, I was involved with the routine activities of the BEST group by attending scheduled group discussions, data meeting and seminar and symposium. I have disseminated part of my research output through attending IEEE sensors conference 2017 and 2018 and European researcher’s nights 2016 and 2017. A couple of my research articles are on the publication process.
The BEND programme develops chitosan bio polymer based platform for wearable devices with high flexibility and conformability with any geometric dimensions. The compatibility of the fabricated platform was confirmed with Human dermal cells (HDF, ATCC® PCS-201-012™) adhesion and proliferation. The cell adhesion ability of the platform was enhanced with its functionalization with Glycine. For in vitro neurite differentiation study a nanoscale thin layers of interdigitated Au electrode were established on the Chi membrane. The Human neuroblastoma cells (SHSY5Y, Sigma Aldrich) were established and electrochemical investigations were performed. The immobilized cell showed positive response through electrically stimulated differentiation.
The outcome of this program is a breakthrough towards developing a comfortable biomedical device avoiding discomforts of currently available wearable/implantable devices. The newly developed metallization protocols on the chitosan based platform open-up the possibilities of establishing sensor circuitry as well as RFID tag for sensing as well as acquiring signals remotely with hand-hold electronic devices. Thus, this biopolymer based platform holds promise for developing comfortable point-of-care devices for the users. Moreover, biocompatibility together with biodegradability and bioresorbability of this developed chitosan based platform is ensuring their application as implantable devices. The confirmability of this platform allows their applications with other biomedical devices like catheter, fiber optics, and endoscope and even with needles. Such biomedical devices will be able to sense bioanalytes during their primary applications. Thus, patient parameters can be monitored during operating or even collecting the specimen from a diseased subject on real time.The developed bendable platform utilized materials of bio-origin and employed the ecofriendly fabrication processes and hence, complies with the green technology
Live cell on chitosan substrate maintained up to 6 weeks providing standard cell culture environment