Microfluidic and Electrokinetic Manipulation of Single Cells

Traditional cell assays report on the average results of a cell population. However, a wide range of new tools are being developed for a fundamental understanding of single cell's functionality. Nonetheless, the current tools are either limited in their throughput or the accuracy of the analysis. One such technology is electrorotation. Although it is known to be unique in its capability for single-cell characterization, it is commonly a slow technique with a processing time of about 30 minutes per cell. For this reason, this thesis focuses on the development of a 3D electrode based electrorotation setup for fast and automatic extraction of a single cell's spectrum. For this purpose, new fabrication processes for 3D electrodes were developed to achieve high-resolution patterning of 3D metal electrodes. The first process we developed was a subtractive one based on passivated silicon structures and the second process was an additive one based on SU-8 photolithography. The additive nature of the second process enables high patterning resolution of electrodes and connection layers, while providing high conductivity thanks to the use of standard metal films. The electrodes have been characterized by different electrical measurements to ensure a proper connection and side-wall exposure. Furthermore, we characterized and compared the sheet resistance of planar and vertical layers. A further microfabrication process was developed for integrating the electrodes into microfluidic channels. The process was designed to enable the use of high numerical aperture lenses; for that purpose, a PDMS-mediated bonding process was engineered to seal the channels with a thin glass coverslip. Moreover, the development of a process to realize microfluidic access holes on the back of the wafer reduces the footprint of the chips and facilitates access for the microscope optics. Finally, a pressure-driven system was used together with the chips to achieve high control of liquid injections and to enable fast and precise flow stop. The combination of such a system, together with the dielectrophoretic forces that can be applied by the 3D electrodes, allows accurate positioning of single cells inside the 3D electrode quadrupole. The particles can then be analyzed by electrorotation. For this purpose, a custom Labview interface was built to coordinate the full setup and to acquire a full electrorotation spectrum in less than 3 minutes

Detecting particles flowing through interdigitated 3D microelectrodes

Counting cells in a large microchannel remains challenging and is particularly critical for in vitro assays, such as cell adhesion assays. This paper addresses this issue, by presenting the development of interdigitated three-dimensional electrodes, which are fabricated around passivated pillarshaped silicon microstructures, to detect particles in a flow. The arrays of micropillars occupy the entire channel height and detect the passage of the particle through their gaps by monitoring changes in the electrical resistance. Impedance measurements were employed in order to characterize the electrical equivalent model of the system and to detect the passage of particles in real-time. Three different geometrical micropillar configurations were evaluated and numerical simulations that supported the experimental activity were used to characterize the sensitive volume in the channel. Moreover, the signal-to-noise-ratio related to the passage of a single particle through an array was plotted as a function of the dimension and num ber of micropillars

Metal-Coated SU-8 Structures for High-Density 3-D Microelectrode Arrays

Electric fields can be effectively used to sense, manipulate, and move particles in lab-on-a-chip devices. Nevertheless, the throughput of such devices is a critical issue, which can be effectively improved by increasing the height of the microchannels. For this purpose, vertical electrodes are needed in order to apply electrical stimuli homogeneously over the full height of the channel. In this paper, we propose different fabrication processes based on a conformal coating of 3-D SU-8 structures with metal layers, defining vertical electrodes in microfluidic channels with high aspect ratio and uniform coating of the vertical sidewalls. We describe two different strategies to achieve the patterning of connection lines inside the gaps of the pillar electrodes--one based on liftoff and the other based on dry film resist. We show how the liftoff approach allows for high connection densities and high resolution of the patterning inside the 3-D electrode arrays. Moreover, we highlight how the dry film process provides an efficient and low-cost alternative when neither high-density patterning nor high resolution is needed. Standard resistive and impedance measurements show high conductivity of the structures whose fabrication process grants standard photolithographic resolution in the definition of the electrode features

3D Integration Technology for Lab-on-a-Chip Applications

A review is presented of advances and challenges in fully integrated systems for personalised medicine applications. One key issue for the commercialisation of such systems is the disposability of the assay-substrate at a low cost. This work adds a new dimension to the integrated circuits technology for lab-on-a-chip systems by employing 3D integration for improved performance and functionality. It is proposed that a disposable biosensing layer can be aligned and temporarily attached to the 3D CMOS stack by the vertical interconnections, and can be replaced after each measurement

Metal-coated silicon micropillars for freestanding 3D-electrode arrays in microchannels

This paper presents a fabrication process for arrays of high-aspect-ratio micropillar electrodes, which are freestanding 3D structures that feature metal sidewalls connected to passivated planar wires. Facing vertical electrodes are considered to be a key solution in microdevice technologies, as they are able to improve the efficiency and accuracy of electrical methods by generating homogeneous electric fields along the height of microfluidic channels. Despite the acknowledged advantages of using vertical microelectrodes, current microfabrication technologies do not allow the manufacture of such structures with the same resolution and versatility as planar electrodes. The present study focused on the fabrication of round and square-shaped silicon pillar arrays exposing metal on their sidewalls, which is decoupled from the substrate by means of a passivation layer. The pillars range in width from 10 μm to 70 μm, with gaps down to 10 μm and a maximum aspect ratio of 5:1. Metal deposition and patterning were revealed to be the critical steps of the process. Deposition was achieved by sputtering, while patterning was performed by photolithography, and the photoresist was applied by spray-coating. The pattern was then transferred into the metal layer by means of dry etching. This new process can be adapted to any metal that is suitable for depositing by sputtering and patterning by dry etching. The presence of the metal layer on the vertical sidewalls was confirmed by SEM imaging combined with EDX analysis. The arrays were then characterized by electrical conductivity measurements and impedance spectroscopy

On‐chip technology for single‐cell arraying, electrorotation‐based analysis and selective release

This paper reports a method for label-free single-cell biophysical analysis of multiple cells trapped in suspension by electrokinetic forces. Tri-dimensional pillar electrodes arranged along the width of a microfluidic chamber define actuators for single cell trapping and selective release by electrokinetic force. Moreover, a rotation can be induced on the cell in combination with a negative DEP force to retain the cell against the flow. The measurement of the rotation speed of the cell as a function of the electric field frequency define an electrorotation spectrum that allows to study the dielectric properties of the cell. The system presented here shows for the first time the simultaneous electrorotation analysis of multiple single cells in separate micro cages that can be selectively addressed to trap and/or release the cells. Chips with 39 micro-actuators of different interelectrode distance were fabricated to study cells with different sizes. The extracted dielectric properties of HeLa, HEK 293, and human immortalized T lymphocytes cells were found in agreements with previous findings. Moreover, the membrane capacitance of M17 neuroblastoma cells was investigated and found to fall in in the range of 7.49 ± 0.39 mF/m2