Multifunctional nanostructures for intracellular delivery and sensing in electrogenic cells
Creators: name: BRUNO, GIULIA
Publisher: name: Universit� degli studi di Genova
Year: 2020

description: Biological studies on in vitro cell cultures are of fundamental importance for investigating cell response to external stimuli, such drugs for specific treatments, or for studying communication between cells. In the electrophysiology field, multielectrode array devices (MEA) are the gold standard for the study of large ensambles of electrogenic cells. Thus, their improvement is a central topic nowadays in neuroscience and cardiology [1]. In the last decades, thanks to the adoption of nanotechnologies, the study of physiological and pathological conditions of electro-active cells in culture have becomes increasingly accurate[2], allowing for monitoring action potentials from many cells simultaneously. 
In fact, nanoscale biomaterials were able to overcome the limitations of previous technologies, paving the way to the development of platforms for interfacing the electrogenic cells at unprecedented spatiotemporal scales. These devices, together with microfluidics, are starting to be used for drug screening and pharmaceutical drug development since they represent a powerful tool for monitoring cell response when cultures are stimulated by target compounds. 
Many pharmaceutical agents, however, including various large molecules (enzymes, proteins, antibodies) and even drug-loaded pharmaceutical nanocarriers, need to be delivered intracellularly to exercise their therapeutic action inside the cytoplasm[3]. Nanoscale electrodes offer individual cell access and non-destructive poration of the cellular membrane enabling high capability in the delivery of biomolecules. Among all the techniques, electroporation have proven encouraging potential as alternative to the carrier mediated methods for molecular delivery into cultured cells[4]. 
In this regard, different groups [5][6][7] exploited the integration of nanostructures with delivering capabilities with single-cell specificity and high throughput in biosensing platforms. These efforts provided powerful tools for advancing applications in therapeutics, diagnostics, and drug discovery, in order to reach an efficient and localized delivery on a chip. 
Despite these new tactics, there is still a critical need for the development of a functional approach that combines recording capabilities of nanostructured biosensors with intracellular delivery. The device should provide for tight contact between cells and electrode so as to enable highly localized delivery and optimal recording of action potentials in order to attain a high degree of prediction for the disease modeling and drug discovery. This ?on-chip? approach will help to gain deeper insight in several bio-related studies and analyses, providing a comprehensive knowledge of the entire cellular dynamics when selectively stimulated by the desired bio-molecules. 

In the first part of this dissertation, a solution will be proposed in order to fill this gap and respond to this need in the biology field. 
In the first chapter, I will describe briefly the principles of action potentials and how neurons and cardiomyocyte are composed, together with the development of electrophysiology and the advent of multielectrode arrays.
In the second chapter, more details about fabrication and cell-electrode system modelling will be explained. In the same chapter, I will explore the development of multielectrode arrays up to the present days, along with the advent of nanotechnologies and the related techniques for improving the previous platforms. The different cell poration techniques will be described in order to reach the best recording capabilities without damaging cells. Electroporation, optoporation and spontaneous poration will be presented and the chosen technique for our application (electroporation) will be reviewed more in detail.
In the third chapter, different methodologies for intracellular delivery will be explained, focusing also on the electroporation technique. A small paragraph about the integration of these techniques on chip will be inserted to illustrate the state of the art of these devices.
The fourth chapter will explicate in details the Microfluidic multielectrode array idea, the approach used in order to fabricate this novel platform from scratch, the experiments carried out to verify its capabilities and the associated results. 

In the last paragraph, I will discuss how the proposed platform could became suitable for the day to day uses in research activity by employing nanoporous materials. 
In fact, big efforts are carried out in order to find appropriate metamaterials as substitutes of the 3D counterparts so as to decrease the cost of device manufacturing that makes them unfitting with research activity. 
As a novel electrode material, nanoporous metals possess unique properties, such as a low fabrication cost, high plasmonic enhancement and large surface-volume ratio[8]. 
Nanoporous gold behaves like a metamaterial whose effective dielectric response can be tuned accordingly to the wanted use. These properties make the material suitable for multiple biosensing application, from a high-performance and reliable SERS (surface enhanced raman scattering) substrate [9] to an electrode in CMOS MEAs capable of intracellular recordings[10]. 
All these properties were explored in the last years, but it could be interesting to further study if the characteristics of this material could make it a good photoelectrical modulating material for eliciting electrogenic cells firing activity. In this way, this technology could be in principle easily implemented on commercial CMOS devices, consenting stimulation and recording at single cell level with high-resolution sensors, opening the way to new methodologies for studying electrogenic cells and tissues. 
Electrical stimulation of excitable cells is the basis for many implantable devices in cardiac treatment and in neurological studies for treating debilitating neurological syndromes. In order to make the technique less invasive, optical stimulation was widely investigated [11]. The non-genetic photostimulation is starting to make its way in the field since it allows to avoid changing the biological framework by using transient thermal or electrochemical outputs from synthetic materials attached to the target cells[12]. If stimulated with impinging light these materials could inject free charges into the solution resulting in an ionic current at the interface able to eliciting of neurons[13] or cardiomyocyte action potentials.
Plasmonic porous materials have all the suitable properties to be considered as an appealing tools for charge injection and consequently for stimulation of electrically active cells [14]. 

Thus, the second part of this dissertation will exploit the capabilities of these plasmonic metamaterials, placing particular emphasis on the possibility of photoelectrochemical modulation.
In particular, in the fifth and last chapter I will describe all the properties and application of the porous material and the mechanism of photoemission. 
In the experimental paragraphs, the free charge photoemission properties of porous gold will be explored together with plasmonic non-genetic photostimulation of the cardiac cells on commercial CMOS MEAs.