Yuxin Cai1,Pengyu Chen1
Auburn University1
Studies of brain activities have been conducted at the frontmost end of neuroscience. The brain is composed of billions of neurons and is in charge of the central nervous system, yet despite the main biological role of the brain, there still are many unknown structures, functions, and connections remaining unknown to us. Meanwhile, the development of medical and clinical research has been phasing in the <i>in vitro</i> inspection of brain cell and neuron-related mechanisms for preclinical studies. Specifically, the <i>in vitro</i> exploration of brain and neuron cell activities in preclinical studies has been conducted to mimic the central nervous system response to advanced new medicines or medical technologies and to assist deliver precision treatment for brain diseases.<br/><br/>Parallel to fluorescence methods, localized surface plasmonic resonances (LSPR) has become a particularly interesting technique for studying the chemistry of living cells. LSPR is a label-free, non-invasive technique, confined to subwavelength-size noble metal nanoparticles that possess large optical cross-sections which have been thoroughly studied. The nanoplasmonic structures propose remarkable potential in sensor sensitivity, tunability, miniaturization, and large-scale fabrication, which makes it possible to intrinsically record neural cell activity at a single neuron level by optical means.<br/><br/>Deciphering the mechanisms of brain and neuron-related activities requires the acquisition of detailed information from a wide range of different scales, ranging from neuron networks to single neuron cells. In our study, we directly measured the change of surface electron density on a gold nanoparticle that is induced by the local neural cell's electric field to monitor brain neural activity. A 1 cm<sup>2</sup> area sensing array of 100 um-high and 5 um-diameter antennas was used, which is large enough to analyze the collective behavior while offering high resolution to observe single neural cell activity.<br/><br/>Because of its low cost, easy processability, biocompatibility, and hydrophobic nature, polystyrene is widely used for biomedical research. First, we designed the desired basal face of the 3D structure and a silicone mode will be manufactured through lithography. By precisely manipulating the etching time, the photoresist layer, and the ultraviolet light source, we are able to control the height and shape of the pattern, and finally formed a micro-antenna array mode for unlimited polydimethylsiloxane (PDMS) microwell masks. Then, a polystyrene solution (20% w/v in toluene) will be cast onto the PDMS mask and covered with indium-doped tin oxide (ITO) coated glass. After degassing, processing, and evaporation of the organic solvent, the base layer of our micro-antenna sensing array will be subsequently coated with gold nanoparticles to serve the sensing purpose with the strong plasmonic coupling can be observed by our previous developed LSPR dark-field imaging technique.<br/><br/>Murine hippocampal cell line (H19-7) and dissociated hippocampal neurons were observed on our micro-antenna sensing array. After a common preparation of the sensing array surface, differentiated hippocampal cells and hippocampal neurons were transferred to the sensing surface and further cultured, forming a neuron-network-like <i>in</i> <i>vitro</i> cultured specimen and later treated with different formulations. Firstly, the baseline was set when there was rarely any scattered light change captured before any formulation was injected. However, after the formulation injection, spiking activities were spotted under the dark field microscope. According to the references, the shape of the signals is typical of results that would be obtained in extracellular electrode recordings. Within the microfluidic channel designed and applied, it will be easy to use the device to acquire real-time <i>in vitro</i> neural cell activity of any formulations of medicine.