Bioimpedance Measurements on Cell Cultures

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Bioimpedance Measurements on Cell Cultures

Fabrication of Microelectrode Systems:

3- and 4-point electrodes

Adam Andrzej Nieweglowski

Thesis submitted for the degree of

Master in Electrical Engineering, Informatics and Technology (Microelectronics and Sensor

Technology) 60 credits

Department of Physics

Faculty of Mathematics and Natural Sciences

UNIVERSITY OF OSLO

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Bioimpedance Measurements on Cell Cultures

Fabrication of Microelectrode Systems:

3- and 4-point electrodes

Adam Andrzej Nieweglowski

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Bioimpedance Measurements on Cell Cultures http://www.duo.uio.no/

Printed: Reprosentralen, University of Oslo

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Preface

First of all I want to thank my supervisors Ørjan, Christin and Steffen for your accessibility and guidance throughout the project. Further I want to thank every person who somehow contributed to this thesis especially André, Sisay and Jie. Thanks to MiNaLab engineers Viktor, Christoph and Halvor for your time and assistance in the lab. Last, but not least I want to thank my family and friends who supported me through my studies.

The project was very interesting to work with, I got the hands on experience with clean-room equipment. Moreover I learned a lot of new concepts and technologies.

Unfortunately due to various delays like laboratory tool maintenance or COVID-19 outbreak I was unable to fully finish my fabrication work and test the electrodes.

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Abstract

This master’s thesis is focused on fabrication of two microelectrode systems for measurements on cell cultures. One electrode system is a 3-point sensing system suited for cyclic voltammetry, whereas the second one is a 4-point sensing system suited for electrical impedance spectroscopy (EIS).

The phenomena of electrical and bioelectrical impedance, as well as the phenomena related to the measurements and instrumentation are addressed in the beginning of this work. Moreover, the fabrication theory of the lift off method is presented in terms of photolithography and physical vapour deposition process.

The design of both systems is dictated by the chip-holder’s geometry.

The chip-holder is a device where the electrode system is assembled and cells can be seeded to conduct the experiment. However, in case of the electrode system for EIS measurements, the geometry of the sensing area is additionally justified by the sensitivity simulation performed in COMSOL Multihpysics.

Photolithography process was done without any major obstacles, however the platinum metallization brought some unexpected results. This problem was solved by switching to gold matallization using a different deposition machine. Another issue was connected to the passivation layer which is required to have fully operational electrode systems. Passivation process requires precise alignment relatively to the already deposited metal. The passivation layer was placed/exposed with a significant offset from the desired position on the electrode chips. Thus the electrodes could not be tested. An important manufacturing aspect which is waste reduction is briefly covered at the end.

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List of Figures

2.1 Example of an uniform conductor. From [9]. . . 5 2.2 Example of a plate capacitor. From [9]. . . 6 2.3 Single-shell model of a single cell in suspension. . . 7 2.4 Helmholtz’ (left) and Gouy-Chapman’s model of electrical

double layer and electric potential as a function of distance from the electrode. From [10]. . . 10 2.5 Equivalent circuit of single-shell model cell adherent on

electrode surface. From [13]. . . 12 2.6 Electrode configurations. . . 13 2.7 Potential waveform used for cyclic voltammetry (CV). From

[14]. . . 14 2.8 A reversible and a quasi-reversible cyclic voltammogram

example. From [14]. . . 15 2.9 Lift-off sequence using positive photoresist. a) Cross-section

view of photoresist coating on substrate’s surface. b) Exposure process with mask applied. c) Substrate with developed photoresist after exposure. d) Metal coating (deposition). e) Substrate after lift-off procedure. . . 17 2.10 Typical resist spinner. From [27]. . . 18 2.11 Grouping of thin film deposition techniques. Re-created

portion from [30]. . . 21 2.12 Flow diagram of PVD process. . . 21 2.13 Sketch of EB-PVD process. High energy electron beam is fo-

cused on the desired material. When the appropriate temperature is reached, given material starts to evaporate. Va- pour particles are transferred through the vacuum (low- pressure) chamber towards the substrate. Vapour condensates on the substrate forming a thin film. . . 22 3.1 Picture of 3-point chip-holder and schematic drawing of

chip-slot. One chip-holder can fit 3 electrode chips. . . 26 3.2 Picture of 4-point chip-holder and schematic drawing of

chip-slot. One chip-holder can fit 3 electrode chips. . . 26 3.3 Preview of 3-point electrode CAD design made with

FreeCAD. All measures are given in mm. Leads are 0.1 mm wide. . . 27

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3.4 A close-up figure of the sensing area of the electrode suited for CV measurements. . . 28 3.5 Full geometry of the well in COMSOL simulation. The

electrodes are placed on the glass covered by the PBS water. 29 3.6 Current density shown at height of 100µm above the elec-

trodes. Electrode position in this specific figure corresponds to the finally chosen position for the design. . . 30 3.7 Combined current density fields. . . 30 3.8 COMSOL Multiphysics simulation results of the first four

electrode configuration coordinates shown in table 3.2. . . . 31 3.9 Simulation result of rectangle with electrode positions (X, Y)

= (1000, 1200). This spacing was used further in design and fabrication process. . . 32 3.10 Preview of 4-point electrode design made with FreeCAD. All

dimensions are given in mm. Leads are 0.1 mm wide. . . 33 3.11 Photoresist coating. Glass wafer placed in a spinning

machine before spinning. . . 34 3.12 Soft bake, 1 minute at 115^oC. Si wafer was used to keep the

glass wafer clean. . . 35 3.13 Substrate placed in uPG501: Mask-less aligner. Small

rectangular tape was used as referance point. . . 36 3.14 Developed pattern after exposure of S1813 photoresist. . . . 37 3.15 Leybold E-beam PVD machine. . . 38 3.16 Developed sample placed on the small stage ready to be

attached on the big sample holder in the deposition chamber. 38 3.17 Gold deposition in Leybold E-beam PVD. Samples shown

before and after the process. . . 39 3.18 Angstrom EBPVD; chamber, crucible carousel with cooling

system and samples before deposition attached on the stage. 41 3.19 Lift-off sequence. (a) Whole wafer covered in gold after the

deposition process. (b) Wafer immersed in the acetone for 10 minutes without any movement. (c) Wafer was then sprayed with acetone, immersed in a clean acetone and placed in an ultrasonic bath for 5 minutes. (d) Spraying with acetone was repeated, wafer was immersed in a clean acetone again and placed in ultrasonic bath for 10 minutes. (e) The wafer was then sprayed with acetone and isopropanol before it was immersed in a clean isopropanol and placed in the ultrasonic bath for 10 minutes. (f) The substrate was taken out, sprayed with isopropanol and dried with nitrogen-gun. . . 43 3.20 SU8-100 applied on the glass wafer placed in the spinning

tool. Before and after the spinning sequence. . . 44 3.21 Glass wafer placed on top of silicon wafer inside of Roffin

Laser Cutter. . . 46 3.22 Finding optimal scribing parameters. A few 10x30 mm rect-

angles were scribed, cracked and tested in the photolithography process. . . 47

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4.1 Gold electrodes after the lift-off sequence. Any major contaminants are out of the electrode-system area. . . 50 4.2 Microscope pictures (magnification x5) of the gold electrodes

after the lift-off sequence. . . 50 4.3 3-point electrode. Profile of thin film (Ti 20 nm and Au 100

nm). Vertical distance measurement≈1300Å. . . 51 4.4 4-point electrode. Profile of thin film (Ti 15 nm and Au 100

nm). Vertical distance measurement≈1100Å. . . 51 4.5 Platinum electrodes after the lift-off sequence. Magnified

cracks and contamination. The biggest and most visible contamination spot on top is out of the electrode-system area. 52 4.6 Microscope pictures (magnification x5) of the platinum

electrodes after the lift-off sequence. . . 53 4.7 Profile of thin film (Ti 20 nm and Pt 100 nm). Vertical

distance measurement≈15000Å. . . 54 4.8 SU8-100 photoresist exposed and developed to provide

passivation layer. As shown in the figure, due to an unexpected alignment issue pattern was exposed in the wrong place. . . 54 4.9 Profile of the SU8-100 layer (spinning settings correspond to

100 µm thickness). Vertical distance measurement ≈ 1.2× 10⁶Å. . . 55 4.10 Fitting of the 4-point electrode chip (without passivation

layer) into the chip-holder. On the left is the final design whereas on the right is the first design which was adjusted based on the observations. Chips had to be 9.95 mm wide to fit correctly into the chip-slot. . . 55 4.11 Electrode chips assembled in the chip-holder. . . 56 4.12 A test of an alternative electrode setup and a horizontal

reference point issue. . . 57 4.13 A more realistic sketch of a lithography and a lift-off process

forming wings. 1 is the substrate, 2 is the resist layer and 3 is the thin film layer. From [41]. . . 59 4.14 An ideal single-layer resist overhang with negative slope

profile. 1 is the substrate and 2 is the photoresist layer. From [41]. . . 60 4.15 Profile of a thin film (Ti 10 nm and Au 50 nm) using bi-layer

photoresist in lithography process. . . 60

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List of Tables

3.1 Relative permittivity used in COMSOL Multiphysics simulation. From [33]. . . 29 3.2 Different electrode positions tested in the sensitivity simu-

lation. The values corresponds to four different positions;

for example (750, 1500) corresponds to all possibilities of coordinates (±750,±1500). . . 31 3.3 Deposition flow of 15nm titanium contact layer using Ley-

bold E-beam PVD. . . 39 3.4 Deposition flow of 100nm gold layer using Leybold E-beam

PVD. . . 40 3.5 Parameters for deposition of 20nm of titanium and 100nm of

platinum. . . 42 3.6 Spinning sequence for SU8-100 to get approximately 100µm

[40]. . . 44 3.7 Laser parameters for glass cutting. Based on findings shown

below in "Parameter Findings" (3.4.5). . . 46

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1 | Introduction

Rapid development in computer science and data analysis in recent years has contributed to quick development in various engineering fields and technologies. Data processing takes an essential role in our every-day life and beyond it. From practical and innovative technologies like Internet of Things [1] to medical application where machine learning is employed more often [2]. In order to gather data, accurate sensors are needed. In medical applications, electrodes provide interfaces to sense signals.

There are a lot of neurological-oriented research projects in which micro- and nanotechnology is required [3]. University of Oslo takes part in a European project called Training4CRM, coordinated/run by the Technical University of Denmark (DTU). Its objective is to increase understanding of neurodegenerative disorders (e.g. Parkinson’s, Huntington’s) and to work towards development of an implantable device for their treatment.

1.1 Motivation and Goals

People affected by neurodegenerative diseases are not only having a hard time functioning in everyday life, but are also an emotional and physical burden for their families and friends. They often cannot continue their careers properly or take on new responsibilities in life.

Depending on the progress of the disease, constant care is usually required. Treatments are often focused on pharmacological solutions.

Non-pharmacological approach is less direct involving e.g. cognitive intervention, psychological therapy, occupational therapy. Alternatives such as art therapy, aromatherapy or virtual reality are also explored [4]

[5].

Furthermore, studies shows that dementia is one of the most costly diseases among brain disorders in Europe [6]. Not only direct healthcare is taken into the account, but also costs of direct and indirect non-medical costs. Intense brain research and clinical trials are needed in order to increase understanding to be able to impact development in this field.

Interdisciplinary global work is oriented to come up with novel drugs and strategies to increase quality of life of affected people and to face economic challenges this brings to the society [7].

Mentioned earlier, Training4CRM is a project where 15 researchers from European universities are working to address gaps in Cell-based Regenerative Medicine (CRM) and developing a treatment option within

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neurodegenerative disorders. Those diseases are characterised by loss of structure, function and/or death of neurons in the brain. Human stem cells (hSCs) have the unique ability of differentiating into any other cell types. The idea is to take advantage of this trait and use it to replace and/or rebuild the tissue structure of damaged cells.

An implantable device providing given treatment is an ultimate aim.

However, to achieve this goal, various biological, electrochemical and technological challenges need to be faced. Impedance and voltammetry measurements are used to monitor and characterise cell cultures, as well as characterise electrodes in terms of the configuration and material used to fabricate them. It is worth mentioning that bioimpedance measurements gained a lot of attention recently because this technique is not invasive to the subject of interest [8]. My work resolves primarily around fabrication process and testing of two electrode systems.

1.2 Structure of This Thesis

This thesis is further divided into three chapters. The following chapter presents the theory background of electrical impedance and bioimpedance in context of cells. Additionally the theory of instrumentation, measurements, materials and fabrication process is presented. In the next chapter the fabrication process is shown, starting with the design and simulation to the clean-room laboratory work done in order to manufacture the electrodes. Finally, in the last chapter the results and possible improvements are presented and discussed.

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2 | Theory

We can consider the biological material, together with sensing electrodes, as a whole system, sort of "black box". The measurement’s objective is to characterize the content of the box without "opening" it. The results help to describe electrical behaviour and understand any physical or chemical processes inducted inside the box by excitation signals. An equivalent circuit is used to model the "black box" behaviour. The model is based on well-known components and there can be more than one model fitting given "black box". Depending on the application, we may desire to distinct between electrode and tissue contributions.

This chapter is covering basic electrical impedance and bioimpedance to set base for understanding of instrumentation theory and measurements techniques. The impedance theory will be followed by description of electrode double layer; one of the important challenges connected to the electrode interface. Measurement techniques and materials are introduced as well. Fabrication theory is presented at the end of this chapter.

2.1 Electrical Impedance

To set base for the description of measurement models we start with background theory of general electric impedance.

2.1.1 Impedance and Admittance

Impedanceis a measure of the opposition (impede - to oppose) to the current flow in the circuit. This can be described by famous Ohm’s law as the ratio of applied voltage (v) and current (i):

Z= ^v

i[_Ω] (2.1)

Ωis the SI derived unit called Ohm.

Admittanceis the inverse of impedance, to admit instead of oppose. This is describing how easily current flows through a material:

Y=Z⁻¹= ⁱ

v[S] (2.2)

The unit of admittance is called Siemens, and is simply the inverse of Ohm, S= _Ω⁻¹_.

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Both of these measures are complex numbers. For impedance, the real component is determined by the circuit’sresistance(R). The imaginary component is called reactance (X) affected by both the inductive or capacitive properties. In Cartesian form we get:

Z= R+jX (2.3)

Similarly for admittance:

Y= ¹

Z =G+jB (2.4)

where G is real part calledconductanceand B is imaginary component called susceptance. Using equations 2.3 and 2.4 we can obtain the relationship between the components R, X, G and B:

G= ^R

R²+X² and B=− ^X

R²+X² (2.5)

R= ^G

G²+B², and X=− ^B

G²+B² (2.6)

this shows that obtaining components of admittance, automatically gives us information about impedance, and vice versa.

Different representations of complex numbers may be used. For impedance we get:

Exponential form: Z= |Z|e⁻^jθ

Polar form: Z= |Z|cos(θ) +j|Z|sin(θ) based on phasor calculations we have the following:

θ =tan⁻¹ X

R

R=|Z|cos(θ)

|Z|=^pR²+X² X =|Z|sin(θ)

For admittance we have to accordingly switch admittance with impedance (Y with Z), conductance with resistance (G with R) and susceptance with reactance (B with X).

2.1.2 Resistivity and Conductivity

Resistivity, ρ, of a given material is defined as the ratio of the magnitudes of electric field (E) and current density (J):

ρ= |_E|

|J| ^(2.7)

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Consider the following conductor

Figure 2.1: Example of an uniform conductor. From [9].

Assuming the magnitudes of current density and electric field are uniform in the entire conductor, the total current is I = J A and potential difference of the end points is given by V = EL. Substituting this into Ohm’s law and combining with definition given in equation 2.7 we get the relationship between resistance and resistivity:

R= ^V I = ^EL

J A = ^ρL

A (2.8)

Conductivityσis a reciprocal of resistivity:

σ= ¹

ρ (2.9)

this gives us relationship between conductance and conductivity:

G= ^σA

L (2.10)

sinceG=1/R.

2.1.3 Reactance

Reactance, the imaginary part of impedance in a circuit, can be described as the opposition to the changes in applied signal. We distinguish between capacitive and inductive reactance. In general, an ideal resistor should not have any reactance, while an ideal capacitor/inductor should not have any resistance contributing to the circuit’s total impedance [9].

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Capacitive reactance (X_C) is the opposition to the change of voltage across an element in a circuit, defined as:

X_C= ¹

ωC (2.11)

whereωis the angular frequency equal to 2πf.

Figure 2.2: Example of a plate capacitor. From [9].

Definition of capacitance is given as follows:

C= ^Q

V_ab[F] (2.12)

unit is called farad. We can rewrite it in terms of geometry of the plate capacitor shown in figure 2.2 and get:

C=eA

d =e_re₀A

d (2.13)

The value is inversely proportional to the distance between the capacitor’s plates and proportional to the area of overlapping plates and the permittivity of the material in between (e). This material is often called dielectric material, or simply dielectric. Thus,e_ris called dielectric constant or relative permittivity (relative to vacuum permittivitye₀, which was introduced by Coulomb in 1785). From Greek, dielectric means material which an electric field penetrates (static electric field does not penetrate conductors), so we can simply call any material applied in between capacitor’s plates a dielectric. As you may see in the next section, dielectrics are a wide topic leading to new phenomena important in bioimpedance theory.

Inductive reactance (XL) does not usually occur in bio-electrical context, so it will not be described in details. It is a measure of opposition of the change of current across an element in a circuit, defined as:

X_L= ωL=2πf L (2.14)

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2.2 Bioimpedance

By introducing dielectrics in the previous section, we enter the bioimpedance world, since bio-materials often represent some of the dielectric properties. Biological tissue is wet material with free ions ready to move. Some important features of wet material, like double-layer formation or polarization (material and electrode) need to be accounted for.

2.2.1 Cell Model

Living tissue is so complex that it is impossible to model it exactly.

Simplified electrical models are required to understand and analyze measurements. Widely accepted in the field, single-shell model of a suspended cell is the simplest representation of biological cell with a lipid bilayer plasma membrane. All three domains of the model have different electrical properties. The cytoplasm and the medium are both conductive.

Whereas membrane acts like an insulator, thus it has dielectric properties.

Figure 2.3: Single-shell model of a single cell in suspension.

2.2.2 Dielectrics: Wet Tissue

As mentioned before, material used in capacitors is often called dielectric material. However, a more precise definition of dielectric is a material in which displacement current is larger than the in-phase current, when ωC> Gor f >σ/2πe. The frequency-dependent definition is not practical in general, so we could also say that a given material is a dielectric if it has the ability to store energy capacitively, and not only dissipate it [10].

From a historical point of view, the theory has evolved from Coulomb’s ideas in the eighteen century through Maxwell and Debye to Cole models

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used nowdays.

2.2.3 Polarization

Starting with static polarization with bound charge carriers will allow us to understand the electrical concepts of living tissues.

Generally speaking polarization is an event where charge distribution in a given volume is displaced/oriented by an electric field. External polarization is called exogenic polarization. Polarization may occur as a result of internal electric field as well, then it is calledendogenicpolarization [10].

We distinguish between three types of polarization:

• Electronic Polarization occurs in atoms or molecules as a result of displacement of negatively charged electrons relatively to nucleus with opposite charge.

• Orientational Polarization comes from permanent dipoles formed by polar molecules (e.g. water which is unsymmetrical). Those dipoles will also be influenced by the external field.

• Ionic Polarization occurs due to displacement of positive ions relative to negative ones.

2.2.4 Relaxation

Polarization is a phenomena which depends on the electric field and is independent of time. It was believed that variations in size and direction of the electric field induces polarization synchronously. However, the system needs time to react, and particle displacement does not happen in sync with changing electric field. This time dependence is described by the concept of relaxation.

Relaxation was first used by Maxwell to describe elastic forces in gases, later used by Debye in terms of time needed for dipoles to orient themselves. Low enough frequency allows particles to rearrange their positions completely and results in maximal polarization. Higher frequencies results in lower polarization and permittivity.

Relaxation is a time-dependent phenomenon and relaxation time is said to be the time it takes for a system to relax to an equilibrium after exposing to a step function excitation signal [10]. However it has an asymptotic form, meaning that in fact it takes infinitely much time to reach the equilibrium completely. Thus it is helpful to use different parameter to describe this phenomenon. Time constant, τ, is the time it takes for the system to reach 1−e⁻¹ ≈ 0.63 of the equilibrium state. In RC circuits it is used to describe charging and discharging of the capacitor.

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2.3 Instrumentation and Measurement Techniques

2.3.1 Electrodes

Electrode provides interface between ionic and electrical current. Depend- ing on interest and application, electrodes vary in sizes and shapes. From relatively big ECG electrodes where current is induced by biological tissue, to micro electrodes used for bothin vitroorin vivoexperiments where current is induced externally to study tissue response or to pick up signal induced by biological the tissue (e.g. neurons or heart cells).

Micro electrodes are desired for measurements on cell cultures. How- ever, multiple issues have to be addressed in order to model and understand the data. Dielectrics properties (discussed in previous chapter) can be employed to model phenomena occurring at the interface of electrode and biological tissue forming double layer.

Another important aspect is the fact that in electrochemistry we say that an electrolytic cell is polarized if current passes through it, thus any current carrying (CC) electrode will be polarized. Sensing electrodes are called pick-up (PU) electrodes.

For CC we distinguish between:

• Equilibrium state. When the applied voltage is too low to induce current flow.

• Overvoltage change. When the applied voltage is increased enough to induce the current flow.

2.3.2 Electrode Double Layer

When two different molecular structures are in contact, rearrangement takes place in the transition zone. Formation of a double layer occurs in the place of friction of two solid metals, generating static electricity when pieces are separated. Solid metal in contact with liquid results in creation of one fixed and one mobile part of the double layer. The fixed one is on the solid side and the mobile one is on the liquid sied. We do not experience formation of a mobile part in the metal side, since the atoms or molecules are strictly bound. The electrode is either a sink or source of electrons, and electron transfer is the phase where electrons exchange charges with ions.

A double layer forms as soon as the metal is wetted and electron transfer takes place somewhere in the double layer [10].

Different models are used to describe the double layer phenomenon, the simplest one was introduced by Helmholtz in 1853 shown in figure 2.4. In this model, charge separation generates an electrical potential which can be described as a molecular plate-capacitor. One plate is the electrode surface and the other is formed by the ions. The distance between "plates" is of molecular size, about 0.5 nm order, thus the capacitance values are very high as the distance appears in the numerator, like in equation (2.13) [11].

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Figure 2.4: Helmholtz’ (left) and Gouy-Chapman’s model of electrical double layer and electric potential as a function of distance from the electrode. From [10].

The capacitance in Helmholtz’ model does not match experimental observations where the capacitance varies with potential. In early 1900, Gouy and Chapman proposed another model (figure 2.4 right). In contrast to Helmholtz’, the potential drop is smoother based on the thermal motion between ions in the double layer and diffuse part of the solution. This potential drop depends on the ionic concentration and electrode potential [11].

An approximation was introduced by Debye and Huckel. However, a more precise and complex model was proposed by Stern combining Helmholtz’ and Gouy-Chapman’s model. To avoid the possibility of very small distances and almost infinite capacitance, Stern’s model takes ion size into account and does not allow ions to get to the electrode surface closer than its own radius. Diffuse layer in this model is divided into inner and outer layer [10] [11].

More models were introduced with further division of the layers and less assumptions providing a more realistic response of the model.

Complexity of the models increases significantly and it depends on the objective of the experiment which one fits best.

2.3.3 Electrical Impedance Spectroscopy (EIS)

Electrical Impedance Spectroscopy (EIS) is an non-invasive and label-free measurement technique where alternating (AC) signal, often sinusoidal, is applied in order to study current-potential response. Let E describe electrode potential, we have:

E(t) =Epolarization+_∆Esin(ωt) (2.15)

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Epolarizationis the base polarization potential [V],∆Eandωare respectively amplitude [V] and frequency [Hz] of applied signal.

The linear response of the system results in sensed signal which has different amplitude and phase:

I_r(t) =_∆I_rsin(ωt+φ) (2.16) where Ir(t) is a response current as a function of time, ∆Ir is current amplitude [A] andφis the phase angle.

If those two signals, E(t) and I(t), are in phase, impedance is simply Ohm’s law with real components only. The phase offset results in additional imaginary parts. Complexity of biological tissues are analyzed using basic impedance theory which was presented at the very beginning of this chapter (e.g. equation (2.1) and (2.3)).

There are a few variations of impedance spectroscopy on cells. One can analyze cells adherent on top of electrode surface, suspended cells in a medium or cells in a flow-system. It is important to notice that all of the techniques are fairly similar and are associated with each other;

for example, when studying adherent cells, impedance of whole medium affects the measurement just like suspended cells monitoring [12].

Adherent Cells

This measurement technique is based on single-shell model presented in section 2.2.1. There are different ways of modeling how current passes through cells, it can be purely mathematical (Giaever and Keese came up with mathematical approximation and impedance equation [12]) or based on an equivalent circuit considering cells as a capacitance, mix of capacitance and resistance (figure 2.5) or by utilizing different components like constant phase element (CPE). Regardless of which model is applied there are a few common features

• Total impedance of the system consists of electrode-solution interface (double layer), solution impedance, cells, cell-to-cell connection, electrodes, wires connecting electrode system to the impedance analyzer. Basically everything between input and output from impedance analyzer.

• Interesting frequency range is between 100 Hz and 100 kHz.

• By subtracting the baseline impedance caused by electrodes, measurement system and cell culture fluid, information about the cells’

impedance can be obtained.

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Figure 2.5: Equivalent circuit of single-shell model cell adherent on electrode surface. From [13].

This measurement technique is widely used in research and in medical applications. Mainly employed in sensors to study biological activity associated with cellular morphology or membrane quality. Two main processes are proliferation and vitality

• Cell proliferation: growing cells (size and number) contribute to increased total impedance

• Cell vitality: impedance can detect cell apoptosis or necrosis which is expressed by membrane integrity as well as cell number

However, impedance monitoring of adherent cells can be used to analyze processes which are important for cancer detection and understanding. Among those processes we find, cell migration, cell metastasis, cell cycle, cell invasion or simply cell adhesion [12].

Suspended Cells

This technique is usually used for long time monitoring of the total impedance (real and imaginary component) of the medium where living cells are always present. Those cells can be for example bacteria and are freely floating in the medium. Single-shell model or other more advanced models can be simplified to single particles. Metabolism processes affect the conductivity of a given medium by releasing ions into it.

There are some variations of this measurement technique, for example magnetoimpedance spectroscopywhere impedance changes are studied under influence of external electromagnetic field [12].

To put it into perspective, for global food industry it is very important to detect foodborne pathogens. These pathogenes are a huge risk for safety and health of humans, animals and plants. Early detection and studies can provide valuable data decreasing this risk.

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Cells in a Flow-System

Staying in the field of bacteria detection; flow-system measurements gained a lot of attention allowing to individually analyze a big amount of cells flowing through microfluidic channels. Therefore, this technique is not only limited to bacteria detection, but is also applied to study human cell lines, phytoplankton or erythrocytes.

Models describing this technique are often based on single-shell cell models, but the equivalent circuits strongly depend on position of the electrodes. Different ideas were implemented over the years, integrating microelectrodes on the walls of flow-channel is often chosen instead of placing electrodes at both sides of the device.

DC signal can be useful to count or to measure the volume of the cells in the channel. However, use of AC signal allows for richer data collection [12].

Configurations and Sensitivity

We can now look at different electrodes configurations

(a) One-port, two-terminal network (b) Two-port, four-terminal network

Figure 2.6: Electrode configurations.

A single electrode is not enough to conduct any experiments. An electrical circuit needs to be closed, therefore at least two electrodes are required. The network shown in figure 2.6a is the simplest system with only one port, it is called two-terminal or simply two point system.

The given port is used both to apply electrical signal and to sense the response. It means that both electrodes have to act as CC and PU electrodes, what results in polarization.

To avoid polarization of sensing electrodes, an additional port is connected forming a four terminal network, shown in figure 2.6b. Inducing current at one port while sensing the differential voltage at the other results in impedance measurements. Ideally, current should not pass through PU-pair now. However negligible amount may be induced as a result of endogenic and exogenic processes.

Transfer impedance and sensitivity strongly depend on the distance between two ports and the size of the electrodes. Sensitivity is calculated with the following equation [10]:

S= ^J¹·J₂

I₁I₂ [1/m⁴] (2.17)

whereJis a current density vector, andIis the current. The dot product is defined as: a·b = |a||b|cos(θ) with θ being the angle between two vectors. Therefore the sensitivity is a scalar value.

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When the two vectors are perpendicular to each other sensitivity will be zero. If the angle is less than 90^o the sensitivity will be positive and negative sensitivity occurs at places whereθ>90^o.

It is important to find a correct geometry corresponding to highest sensitivity in a model and to avoid or minimize the negative sensitivity.

Negative sensitivity flips the impedance measurements; when the insulator (high impedance) is placed in the negative sensitivity field its contribution to the total impedance is reversed, meaning that the total impedance of the system will decrease. Vice-versa for the good conductor with very low impedance, when it is placed in the negative sensitivity field the total impedance of the system will be higher [10].

2.3.4 Cyclic Voltammetry

Cyclic Voltammetry (CV) is one of the most important electrochemistry measurements where potential-current response is of interest. In this technique redox reactions of electrode-solution interfaces are studied.

Working electrode (WE) potential is ramped up linearly versus time to a predetermined voltage level (forward scan). Then, it is ramped down (reverse scan) resulting in a triangular waveform shown below.

Figure 2.7: Potential waveform used for cyclic voltammetry (CV). From [14].

Usually, start and end potential (pointaandc) is the same but it does not necessarily need to be. The potential is monitored between WE and reference electrode (RE), while current measurement takes place between WE and counter electrode (CE). More than one cycle can be applied, but usually the recorded current curve is different for each cycle [10] [11] [15]

[14].

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(a) Cyclic voltammogram recorded for Fe⁺²/Fe⁺³in aqueous solution

(b) Quasi-reversible cyclic voltam- mograms.

Figure 2.8: A reversible and a quasi-reversible cyclic voltammogram example. From [14].

Behaviour of potential-current response highly depend on the solute and the material used to fabricate electrodes. The figure 2.8a shows an example of cyclic voltammogram of a reversible reaction. It was recorded for Fe⁺²/Fe⁺³ in aqueous solution. During the forward scan electrode- solution reaction results in some products and bi-products which can be then detected during the reverse scan. In a reversible reaction, when the potential sweep is reversed system moves through equilibrium and the current flows from the solution to the electrode converting Fe⁺² back to Fe⁺³.

In contrast to quasi reversible reaction shown in 2.8b the voltammogram peaks should not change their position relative to each other with varying scan rate. A fully reversible reaction characterises by the equal current peaks and fixed potential peak separation

Ep= E^c_p−E^a_p =58/n[mV]

wherenis the number of electrons transferred per molecule.

Quasi-reversible and irreversible processes are strongly dependent on scan rate (V/s); increased scan rate results in increased distance between peaks due to electrode kinetics limitations [11].

Configuration

To correctly interpret acquired data, it is important that electrodes suited for CV have a given configuration and shape. 2D configuration used in [16] shows electrode-system with circular WE in the middle enclosed by CE and RE. Where RE is the smallest one and WE usually has a radius of order of magnitude of 1 mm. This configuration is shown in 3.4.

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2.4 Materials

2.4.1 Chip/Base Material

Substrate material depends on the application and specific needs one can have. Two most common materials, which are biocompatible are silicon and glass wafers.

Silicon is very popular in the semiconductor field. Therefore, in contrast to glass, it is more suitable when electrodes are integrated in a compact electronic device. On the other hand, glass substrate, due to its transparency is compatible for most microscopy systems and therefore may be a huge advantage in research measurements compared with silicon which has to be inspected in different ways not allowing for observations under a regular transmission microscope.

2.4.2 Biocompatible Metals for Electrodes

Electrodes used with biological tissue have to be biocompatible not to impact the object in an unwanted way and to acquire meaningful data.

Common materials for electrodes used on cells are platinum (Pt) and gold (Au) because of good biochemical performance, mechanical properties or ease of modification. Some other materials such as silver chloride electrode (Ag/AgCl), indium tin oxide (ITO), nickel (Ni) or ultra- nanocrystalline diamond can be used depending on needs and specific application [12].

When focusing on Pt and Au¹ it is worth mentioning that these noble metals have poor adhesion to the glass surface. To solve this problem and increase adhesion, an interlayer of either Chromium (Cr), Titanium (Ti) or Alumina (Al₂O₃) is used [17]. Ti could also be a good alternative for the main-metal as it is biocompatible and relatively easy to fabricate with, however reproducibility is low as the material undergoes chemical changes and has a thin oxidation layer.

Platinum is very common in voltage measurements. However, when it comes to amperometric measurements it is not a common choice since it is sensitive to hydrogen present in the solution. The platinum electrode acts like a catalyst for the hydrogen reaction which can produce more current than the subject analyte which is a huge disadvantage for the use as a working electrode. Platinum can still be used in CV measurements but it needs some chemical modifications to improve electron exchange and reversibility of redox reaction. Gold electrodes are preferred in dopamine detection using CV measurements [18–21].

2.5 Nanotechnology and Microfabrication

Micro and nano-fabrication is often oriented very much towards semiconductor industry [22]. Precise thin film pattering can be used to manufacture

1Used in fabrication process in this project

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variety of devices such as gas sensors [23] [24] or pressure sensors [25] and not only being limited to integrated circuits fabrication.

Different techniques are used for electrode fabrication. In this project, lift-off technique has been used. This is an additive method which consists of several steps. Figure 2.9 shows the flow of this method:

Figure 2.9: Lift-off sequence using positive photoresist. a) Cross-section view of photoresist coating on substrate’s surface. b) Exposure process with mask applied. c) Substrate with developed photoresist after exposure. d) Metal coating (deposition). e) Substrate after lift-off procedure.

We can group this into left and right side. Figures from (a) to (c) qualify into photolithography process. Where figure (d) and (e) shows deposition and lift-off procedures.

2.5.1 Photolithography

Photolitography also called optical lithography is widely used in electronics manufacturing. It originates from lithography in which chemical processes make it possible to adhere ink on a smooth surface to print text or artwork. In optical lithography ultraviolet (UV) light is used to make mi- croscopic geometric patterns in a thin film of photoresist on a substrate’s surface. In semiconductor technology silicon wafer is often used, but the material varies with application of the device/component [22].

Wafer Preparation

All lithography procedures take place in a specially designed, enclosed area of a clean room. Conditions are controlled with respect to airborne particles, temperature, humidity, air pressure, vibration and light. Dust particles are unfortunately inevitable and can impact the process in a negative way. Inspection and cleaning of the wafer is needed to maximize repeatability and decrease possibility of error.

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There is a variety of cleaning techniques among which acetone- methanol-ispopropanol sequence is one of the most common in the laboratories. Acetone is a solvent which is primarily used to wash away contamination. It can effectively soften or dissolve organic residues and/or particles, including photoresist. However it leaves an organic film on the surface, therefore washing with alcohol is applied right after:

methanol to dissolve drying acetone and remove thin organic layer, and then isopropanol (IPA) to rinse the surface and remove methanol. IPA is very hygroscopic what results in a dehydrated surface after this final rinse.

One should never let any liquid dry directly on the substrate’s surface.

Every container even in the cleanest lab contains some contaminations to some extend. Evaporating liquids will deposit dust particles on the surface.

Drying with pressurized nitrogen stream is required, it is best to start in the middle of a given sample and work outwards [26].

Photoresist Coating

Photoresist is a liquid, organic polymer which, depending on its properties, reacts differently to exposure to UV light. We distinguish between positive and negative resist. Inpositive photoresist, chemical bindings are weakened up by the light making those spots easier to dissolve. The opposite happens innegative photoresist, where UV hardness the material which becomes less soluble.

Photoresist needs to get evenly distributed on substrate’s surface. There are several techniques (e.g. spray coating or plasma-deposited resist), however spin coating is one of most commonly used. The resist is evenly spread with centrifugal force. A sketch of a typical spinner is shown below

Figure 2.10: Typical resist spinner. From [27].

Not every spinner is equipped with a resist dispenser, then manual application is required. The substrate is placed on the stage where the

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vacuum chuck is keeping it still while spinning. Sometimes, especially when applying thick photoresist, dispensing rotation is used. Around 500 rpm helps to spread the compound slightly, before accelerating to the desired speed which usually is in range of 1000-7000 rpm.

Final thickness of the coating film depends on its viscosity and rotation speed. Thickness can be predicted by empirical equations, and datasheets of a specific photoresist should include graphs which relates final thickness and spinning speed. After spinning, photoresist coating should be uniformly thick and must be chemically isotropic so that its response to exposure and development is uniform. A minimum of 30 second is required to spread resist uniformly [27].

After the coating procedure,soft bake(sometimes called pre-exposure bake or simply pre bake) is needed to evaporate the rest of solvents and build-in stress. This promotes adhesion of the resist onto the surface as well. It is done in a convection oven; 20 minutes at 90-100^oC, or by using a hotplate at around 90-120^oC for 1-3 minutes.

This step is critical for the quality of resist layer. Not sufficiently removed solvent will affect the resist profile. On the other hand, excessive baking may reduce sensitivity and destroy the photo-active compound.

During this step, the thickness is reduced by 10-20%. Using a hotplate instead of convection oven increases control, is faster and does not trap particles.

Exposure

After resist coating and soft bake it is time for exposure. Substrate is carefully placed in the exposure tool where alignment of the mask takes place. Accurate alignment is crucial especially when working with very small geometries using two or more layers.

Every exposure system is equipped with a UV source. Either in form of a focused light beam (mask-less system) where the pattern is "painted"

with movement of the stage under the light source. Another option is an illumination source where light is blocked by a physical mask as shown in figure 2.9b. The smallest features that can be transferred in projection lithography are of the size of the wavelength of the light used. Some techniques, like Resolution Enhancement Technologies (RETs) where mask modification compensates for lithography limitations, allows to go beyond Rayleigh diffraction limit [27]. Used wavelengths:

Extreme UV : 10−14 nm Deep UV : 150−300 nm

Near UV : 350−500 nm

Every resist requires a specific exposure energy. Incident energy, also called dose is given in J/cm²and is dependent on photoresist’s thickness.

This has to be matched with the energy delivered from the machine, by adjusting the exposure time and/or incident light intensity (given in

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mW/cm²). The following equation shows how the three components relate:

Exposure time[s] = ^Dose[mJ/cm²]

Intensity[mW/cm²] ^(2.18) Development

The next step in the process is development. As shown in figure 2.9c, in case of positive photoresist, exposed spots are getting dissolved leaving the desired pattern. This creates a base for further processing using additive or subtractive methods like lift-off or etching. Wet and dry development techniques are used.

Developers and photoresists groups are usually matched together.

Organic solutions are suited for development of negative photoresists, and aqueous alkaline solutions for positive ones. Some developers are preferred in some fields, to manufacture metal oxide semiconductor (MOS) metal ion free developer is recommended. Those developers are based on tetramethylammonium hydroxide (TMAH) [27].

Developers have different dilution properties which may affect development time together with thickness and type of photoresist.

Summary

The whole process can be summarized as follows:

• Wafer preparation. One should inspect the wafer before using it for photolithography. Wafer should be as clean as possible to achieve high quality results and high level of reproducibility. If required, acetone, methanol and isopropanol sequence can be used.

• Photoresist coatingis prerequisite (in additive methods) for transfer- ring any pattern on the surface of the wafer. A thin layer of compound is uniformly spread using centrifugal force under spinning in specially designed device. Coating is followed by soft bake to evaporate excess liquids.

Two photoresists are avaliable. Positive resist gets more soluble after exposure to the UV light. Negative photoresist acts in the other way around.

• Exposure. UV light is exposing the desired pattern in photoresist layer. Light intensity and exposure time is adjusted according to required dose for applied photoresist. It can be performed using mask or in a mask-less device where UV light in form of a beam exposes given geometry. Alignment of several layers is possible.

• Development is needed as a final step to wash off unwanted photoresist. After development, the sample should be inspected and if needed, more development time should be added.

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2.5.2 Thin Film Deposition

Thin film coating is used in a wide range of applications, and is not only limited to electronics. Its applications ranging from space to underwater where thin film coating can be used to increase reliability of sensors or other components to protect them from high pressure or high temperature in their environment [28]. There are a lot of different thin-film deposition techniques which are suited for different application depending on their parameters, like temperature, type of bonding or deposition rate [29].

Figure 2.11: Grouping of thin film deposition techniques. Re-created portion from [30].

Both, Physical Vapor Deposition (PVD) and Chemical Vapor Deposition (CVD) are gaseous processes where vapor transports through low pressure environment or vacuum to result in a thin film on a substrate. The difference lays in the vapor form. PVD is an atomistic process, meaning that material evaporates in form of atoms or molecules from a solid/liquid source to deposit on a substrate’s surface due to condensation (physical process). In CVD vapor undergoes a chemical reaction at the substrate’s surface and results in a thin film [30]. Figure below shows a simple flow of the PVD process:

Figure 2.12: Flow diagram of PVD process.

PVD is usually used for coatings where the thickness is in range of a few nanometers even to a few micrometers [31]. This method can be further divided by the method of vaporization

• Thermal (or vacuum) vaporization. Atoms from thermal evapora- tion are transferred to the substrate’s surface with minimal or no col- lision with gas molecules in between melting material and substrate.

This path is called "line-of-sight".

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• Sputter deposition. This process does not use thermal vaporization.

Instead, particle bombarding and momentum transfer is used to eject surface atoms from the source material. The distance between source material and deposition target is smaller than in vacuum method.

• Arc vapor deposition. High current and low voltage electric arc is used to melt and evaporate cathodic or anodic electrode.

To start vacuum/thermal deposition, the pressure in the chamber needs to be decreased to 10⁻⁵ - 10⁻⁹ torr. The lower the pressure, the higher the deposition quality in terms of contaminations. To conduct the process, source material is heated to a temperature which corresponds to appropriate vapor pressure. Various heating techniques are employed among which we find resistive heating, high and low energy electron beam or induction heating. Every heating method is different in terms of temperature limit, cost and efficiency [31].

Electron Beam Physical Vapor Deposition (EB-PVD)

This technique utilizes a high energy electron beam to heat source material enough to start phase transition. Focused e-beam is delivered by a straight or deflected electron gun. Deflection is often greater than 180^o to protect filament from depositing material. Sketch of the process’ set-up is shown below:

Figure 2.13: Sketch of EB-PVD process. High energy electron beam is focused on the desired material. When the appropriate temperature is reached, given material starts to evaporate. Vapour particles are transferred through the vacuum (low-pressure) chamber towards the substrate. Vapour condensates on the substrate forming a thin film.

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Thermoionic-emitting filament is used to liberate electrons. High voltage (range of 7-20 kV) accelerates particles through magnetic and/or electric field used to focus and direct the beam towards the material. The material is placed in a water-cooled copper crucible, often on a "carousel"

which is making deposition of multiple materials possible without venting the chamber. To ensure even melting and vaporization, the focused e-beam is moved around the surface not to melt through the volume in one spot only. When heating solid insulating materials or dielectrics, charge buildup can lead to arcing and additional contaminations. Electron guns used for the process usually operates with power in the range of 50 kW. However, higher power is sometimes needed [31].

Substrates are mounted above the evaporating material facing it. To control the process and keep deposition uniform, the sample holder rotates, a shutter physically stops the vapor when required and various sensors are used to read the temperature, deposition rate and thickness.

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3 | Fabrication

Laboratory work done in order to fabricate electrodes ready to be used in cell cultures measurements is presented in this chapter. Starting with design and its geometry motivation in form of chip-holder and sensitivity simulation for 4-point electrodes suited for EIS measurements. Proceeding with description of clean-room work in order to perform lift-off method and glass cutting using laser.

Disclousure

The Research Council of Norway is acknowledged for the support to the Norwegian Micro- and Nano-Fabrication Facility, NorFab, project number 245963/F50.

3.1 Chip-Holder

Two different chip-holders were manufactured in cooperation with mechanical workshop at University of Oslo (based on a previous design from Arto Heiskanen, Technical University of Denmark, DTU Bioengineering).

One suited for 3-point contact and the other one for 4-point sensing. The bottom part has three rectangular (10x30 mm) chip-slots. The top part has a well through which cells are adhered on the electrode surface. The well is circular changing into rectangular cube with total volume of around 500µL.

The top-part is also equipped with special slot for a PCB with springloaded pins to interface electrodes with the impedance analyzer. There are three or four holes where pins are placed to make contact with the electrodes.

The figures below show pictures of assembled chip-holders and geometry of chip-slot with measures of projected contact holes and the well. Electrodes were designed according to these measures.

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(a) Empty, assembled chip-holder. (b) Chip-slot, all measures inmm.

Figure 3.1: Picture of 3-point chip-holder and schematic drawing of chip- slot. One chip-holder can fit 3 electrode chips.

(a) Empty, assembled chip-holder. (b) Chip-slot, all measures inmm.

Figure 3.2: Picture of 4-point chip-holder and schematic drawing of chip- slot. One chip-holder can fit 3 electrode chips.

3.2 Design of 3-Point Electrode System

The geometry of 3-point electrode is based on previously fabricated electrodes used in the project. Position of the electrodes and the contact pads relatively to the 10x30 mm chip is dictated by projected position of the well and contact holes onto the chip-slot in the bottom part shown in figure 3.1b.

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The technical drawing with measures is shown below. A small cross was added in each corner, to indicate size of the chip and work as reference points in cutting process later on. However the main application of these 4 crosses is to provide reference points for alignment process under the exposure of passivation layer to cover the leads to avoid electromagnetic field from unwanted spots which are present in the experimental well.

Figure 3.3: Preview of 3-point electrode CAD design made with FreeCAD.

All measures are given in mm. Leads are 0.1 mm wide.

The contact-pads are 3x2 mm rectangles, leads are 0.1 mm wide. The radius of the working electrode is 0.3 mm. The total area of the sensing area with WE, CE and RE is about 1 mm². A close-up picture of the sensing

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area of the electrode is shown below. This particular design is based on a previous design from the Training4CRM project [32].

Figure 3.4: A close-up figure of the sensing area of the electrode suited for CV measurements.

3.3 Design of EIS 4-point Electrode System

As described previously in the theory, section 2.3.3 there are a lot of variations of electrode-setup. One should arrange electrodes such that the area of interest and the sensitivity is maximized. In case of measurements on cells in a circular well, rectangular setup seems to be a good configuration to achieve that. Simulation of electric field in COMSOL Multiphysics was performed in order to maximize the sensitivity by adjusting the position of the electrodes. Some simplifications were made in the simulation model. CAD design was then based on the simulation results.

3.3.1 Sensitivity Simulation with COMSOL Multiphysics

The well was simulated in order to match the real experiment as much as possible. Using parameters of real materials or close approximations. The model was build with circular domains, geometry is shown below. The shape of the well is modeled to be only circular (in reality, the well is both circular and rectangular), however height was adjusted such that volume is 500µL.

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Figure 3.5: Full geometry of the well in COMSOL simulation. The electrodes are placed on the glass covered by the PBS water.

Four circular electrodes were placed on top of a bigger circle which represents glass surface/chip. The tallest cylinder on top of the electrode chip represents the solution in the well. This solution was modeled as PBS water. When adding material in the simulation, relative permittivity,e_rhad to be entered in order to run the simulation successfully. Values are shown in the table 3.1. This is only an estimation since the values often vary with frequency and temperature [33].

e_r

BPS Water 70

Glass 4.2

Electrodes (Pt/Au) 1

Table 3.1: Relative permittivity used in COMSOL Multiphysics simulation.

From [33].

Simulation results

Electromagnetic simulation was performed. First, one pair was passive and one pair was active, then the pairs were switched as described in theory.

Secondly, current density of the electric field was extracted and combined together to calculate the total sensitivity of the system.

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(a) Left pair active, right passive.

Resulting in current density data-set, J1

(b) Left pair active, right passive.

Resulting in current density data-set, J2

Figure 3.6: Current density shown at height of 100µm above the electrodes.

Electrode position in this specific figure corresponds to the finally chosen position for the design.

The two current density fields were combined together:

Figure 3.7: Combined current density fields.

To find out the optimal position of electrodes, the sensitivity equation (2.17) was implemented and result is visualized with a color plot.

The simulation was performed with a few combinations of x and y position relatively to common origin in the middle of the electrodes as shown in figure above. For example, position (X,Y) = (100, 100) gives a square where first electrode has coordinates (-100, 100), second (100, 100) etc. Following table shows tested positions:

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X [µm] Y [µm]

750 1500

1500 1500 1200 1200 1000 1400 1000 1200

Table 3.2: Different electrode positions tested in the sensitivity simulation.

The values corresponds to four different positions; for example (750, 1500) corresponds to all possibilities of coordinates (±750,±1500).

Sensitivity was represented by color-plot for each position pair. Sensit- ivity intensity was judged visually

(a) A rectangular configuration. (X, Y) = (750, 1500).

(b) A square configuration. (X, Y) = (1500, 1500).

(c) A square configuration. (X, Y) = (1200, 1200).

(d) A rectangular configuration. (X, Y) = (1000, 1400).

Figure 3.8: COMSOL Multiphysics simulation results of the first four electrode configuration coordinates shown in table 3.2.

We notice that square setup (figure 3.8b and 3.8c) results in weakest sensitivity, whereas a rectangular configuration resulted in increased intensity. Figure 3.8d shows setup with (X=1000, Y=1400). It has a relatively big sensing area with decent sensitivity, however I decided to use different geometry (X=1000, Y=1200) which gave slightly better impression with a good balance between positive and negative sensitivity:

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Figure 3.9: Simulation result of rectangle with electrode positions (X, Y)

= (1000, 1200). This spacing was used further in design and fabrication process.

3.3.2 CAD Design

The optimal electrode position determined by the COMSOL simulation of the sensitivity field was implemented in the design of whole electrode system. Contact pads and electrodes were placed such that they will align with opening holes in the top part of the chip-holder.

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Figure 3.10: Preview of 4-point electrode design made with FreeCAD. All dimensions are given in mm. Leads are 0.1 mm wide.

3.4 Clean-Room Procedures

Photolithography, metal deposition, lift-off process and laser cutting is presented in this section.

3.4.1 Photolithography

Lithography process starts with wafer preparation. I have washed wafers using acetone and isopropanol sequence as described previously in theory chapter. I was curious if washing gave desired results so washed substrates were compared with unwashed wafers.

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On average, the unwashed samples made a better impression with less spots including particles. Thus the unwashed substrates were used in fabrication process.

Glass Wafer

Double side polished Borofloat 33 glass wafers were ordered fromUniver- sity Wafer[34]. This glass type is a borosilicate glass containing a melted mix of boron trioxide (B203), silica sand (quartz related), sodium carbonate (Na₂CO₃) and alumina (Al₂O₃) [35]. It is known for having very low coef- ficients of thermal expansion, what makes them more resistant to thermal shock than any other commonly used glass [36]. This is an advantage when using laser to cut/scribe the glass at the end of the fabrication process.

Photoresist Coating

S1813 photoresist was used. Following pictures show wafer without and with applied photoresist before spinning.

(a) Glass wafer placed on a stage in a spin coating machine. Cross in the middle is vacuum chuck.

(b) Photoresist added on the surface, before spinning.

Figure 3.11: Photoresist coating. Glass wafer placed in a spinning machine before spinning.

Spinning settings are shown below:

• Spinning time: 30 seconds

• Spinning speed: 3000 rpm

Since resist S1813 is fairly thin, dispensing spin was not applied.

According to the datasheet [37], this rotation speed results in approximate final thickness of 15000Å = 1500nm. Following the spin, soft bake was performed, 1 minute at 115^oC. Chosen temperature is 5^oC higher than the suggested temperature in the datasheet since the wafer was placed on top of Si wafer to keep the main wafer clean as shown in the following picture.

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Figure 3.12: Soft bake, 1 minute at 115^oC. Si wafer was used to keep the glass wafer clean.

Exposure: Mask-less Aligner

Heidelberg instruments uPG501 Mask-less Aligner was used. This tool is better to do quick prototypes without need of ordering a mask. Mask file in form of CAD design needs to be converted into a specific file extension.

This was done by following the steps in the datasheet [38].

Next, sample was carefully placed on the stage using reference tape shown in figure 3.13 (not an original reference point, but used by experienced users at the lab). One could also use alignment pins to make sure the position was the same every time. It is important, because center of the substrate is found by edge detection. Since the wafer is circular, even small changes in the positioning on the stage may lead to much different center point.

Vacuum holes provides suction such that sample is kept still under the exposure.

Bioimpedance Measurements on Cell Cultures

Bioimpedance Measurements on Cell Cultures

Fabrication of Microelectrode Systems:

3- and 4-point electrodes

Adam Andrzej Nieweglowski

Thesis submitted for the degree of

Master in Electrical Engineering, Informatics and Technology (Microelectronics and Sensor

Technology) 60 credits

Department of Physics

Faculty of Mathematics and Natural Sciences

UNIVERSITY OF OSLO

Bioimpedance Measurements on Cell Cultures

Fabrication of Microelectrode Systems:

3- and 4-point electrodes

Adam Andrzej Nieweglowski

Preface

Abstract

Contents

List of Figures

List of Tables

1 | Introduction

1.1 Motivation and Goals

1.2 Structure of This Thesis

2 | Theory

2.1 Electrical Impedance

2.2 Bioimpedance

2.3 Instrumentation and Measurement Techniques

2.4 Materials

2.5 Nanotechnology and Microfabrication

3 | Fabrication

3.1 Chip-Holder

3.2 Design of 3-Point Electrode System

3.3 Design of EIS 4-point Electrode System

3.4 Clean-Room Procedures