1. 1
VINA FARAMARZI
ELECTRICAL – OPTICAL DETECTION
OF UNIQUE MOLECULES
Faculty of Physics
Institut de Physique et Chimie des Matériaux de Strasbourg
(IPCMS)
Master of Condensed Matter and Nanophysics

2. 2
Faculty of Physics
Master of Condensed Matter and Nanophysics
VINA FARAMARZI
ELECTRICAL – OPTICAL DETECTION OF
UNIQUE MOLECULES
Supervisor:
Professor Bernard DOUDIN
Co-advisor:
Dr. Sébastien HARLEPP
June 2007

3. 3
ACKNOWLEDGMENTS
My special thanks to my thesis advisor, Prof. Bernard Doudin for providing me with the
opportunity to accomplish this fascinating thesis in his group.
I would also like to warmly thank my co-advisor, Dr. Sebastian Harlepp for helping me in doing
the optical measurements and analyzing the data.
I would like to dedicate this thesis to my family.

4. 4
Contents:
1 Introduction..............................................................................................................................6
1.1 Molecular Electronics: .....................................................................................................6
1.2 Single Molecule Detection in Junctions2, 3
.......................................................................7
1.2.1 Single Molecule Electrical Detection.......................................................................7
1.2.2 Single Molecule Optical Detection ..........................................................................8
1.2.3 Single Molecule Optical Detection Limits...............................................................9
1.3 Planned experiment..........................................................................................................9
1.3.1 Outlook.....................................................................................................................9
2 Total Internal Reflection Fluorescence Microscopy (TIRFM)13
............................................10
3 Fluorescent probe ...................................................................................................................12
3.1 Fluorescein Isothiocyanate.............................................................................................13
3.2 Bleaching of FITC17, 18
...................................................................................................15
4 Microfluidics:.........................................................................................................................16
4.1 Introduction....................................................................................................................16
4.2 Single Molecule detection in microfluidic channels19
...................................................16
4.3 Microfluidic devices.......................................................................................................17
4.3.1 PDMS (polydimethyl siloxane)..............................................................................17
4.3.2 Fabrication of microfluidic channels: Soft Lithography22
.....................................17
5 Sample preparation:................................................................................................................19
5.1 Sputtering .......................................................................................................................19
5.2 Microfabrication process in clean room environment....................................................19
• Cleaning .........................................................................................................................20
• Spin Coating...................................................................................................................20
• Baking ............................................................................................................................20
• Optical Lithography .......................................................................................................20
• Photoresist developing ...................................................................................................21
• Checking filter................................................................................................................21
• Final Etching Process.....................................................................................................22
• Photolithography for windows of photoresist................................................................22
5.3 Microanalysis and microfabrication...............................................................................23
5.3.1 Focused Ion Beam (FIB)........................................................................................23
5.3.2 FEI Strata 235 Dual Beam .....................................................................................23
6 Device preparation .................................................................................................................24
6.1 Bonding between PDMS and passivated silicon23
.........................................................24
6.2 Wire Bonding.................................................................................................................25
6.3 Electroplating .................................................................................................................25
7 Optical Measurements............................................................................................................26
7.1 Detection of fluorescent Beads ......................................................................................27
7.2 Fluorescent Probes .........................................................................................................28
7.3 Final observation............................................................................................................29
8 Results and discussion............................................................................................................31
9 Conclusion..............................................................................................................................33
10 References ..........................................................................................................................34

5. 5
Figures:
Figure 4-1 - Schema of the excited fluorophores with the evanescent wave.................................11
Figure 4-2 – Inverted microscope configuration for TIRFM.........................................................11
Figure 5-1 – A four-state molecular switch (A=absorbance, λ =wavelength) ..............................12
Figure 5-2 - the chemical structure of Fluorescein, ethanol...........................................................14
Figure 5-3 - the absorption and fluorescence emission spectra .....................................................14
Figure 5-4 - the fluorescence emission spectrum of Fluorescein dissolved in ethanol..................15
Figure 5-5 - Fluorescein Isothiocyanate.........................................................................................15
Figure 6-1 – mould containing the pattern of the microfluidic network........................................18
Figure 6-2 – curing the PDMS over the patterned mould to form the channels, soft lithography.18
Figure 7-1 - Spincoating.................................................................................................................20
Figure 7-2 – The design of positive mask containing the junctions...............................................21
Figure 7-3 – Gold patterns on coverslip.........................................................................................22
Figure 7-4 – before (a) and after (b) the FIB cut............................................................................24
Figure 7-5 – SEM image of the junction with 79.5 nm gap obtained in FIB cut...........................24
Figure 8-1 – Sample with bonded PDMS and inserted tubes to guide the liquid ..........................25
Figure 8-2 - Electrolytic cell for electroplating..............................................................................26
Figure 9-1 – Fluorescent beads (diameter =1 mµ ) detected by TIRF ............................................27
Figure 9-2 – Bleaching process of FITC obtained by TIRFM.......................................................28
Figure 9-3 – Bleaching process of FITC obtained by TIRFM, very low background signal ........28
Figure 9-4 – left: PLAPON 60XTIRFM objective, right: sample holder in TIRFM setup ...........29
Figure 9-5 – Sample placed in a drilled Petri dish.........................................................................29
Figure 9-6 – transmission image of fluorophores over the coverslip containing gold patterns.....30
Figure 9-7 TIRF detection..............................................................................................................30
Figure 9-8 TIRF detection..............................................................................................................31

6. 6
1 Introduction
1.1 Molecular Electronics:
Since the size of components in integrated circuits is shrinking in accordance with Moore’s law,
it is a simple matter to suggest that the ultimate integrated circuits will be constructed at the
molecular or atomic level 1
. Such a scenario was suggested in a 1959 lecture by the eminent
physicist and visionary, Richard Feynman:
“I don’t know how to do this on a small scale in a practical way, but I do know that computing
machines are very large; they fill rooms. Why can’t we make them very small, make them of little
wires, little elements–and by little, I mean little. For instance, the wires should be 10 or 100
atoms in diameter, and the circuits should be a few thousand angstroms across...there is plenty of
room to make them smaller. There is nothing that I can see in the physical laws that says the
computer elements cannot be made enormously smaller than they are now. In fact, there may be
certain advantages.”
Even considering further advancements in the science of making integrated circuits, the fact
remains that the materials and processes currently in use will reach their fundamental limits
sometime in the near future. That eventuality forces us to consider other options. Instead of
continuing to make the existing components smaller and smaller, much research is focused
towards going directly to the smallest components that are likely to be functional– single
molecules and small groups of molecules.
Molecular electronics is, relatively speaking, a young field which has become very active in the
last decade. It’s a technology facing area of science. Its remarkable growth in the last two
decades is a direct reflection of the synthetic capabilities arising from surface fictionalization and
bonding at interfaces, and of the invention of the scanning probe microscopes that permit both
manipulation and measurement at the nanoscale. Even so, there have been many significant
advances and a much greater understanding of the types of materials that will be useful in
molecular electronics, and their properties.1

7. 7
1.2 Single Molecule Detection in Junctions2, 3
Single-molecule detection is of fundamental and practical interest because the behavior of the
finest constituents of matter can be observed and it’s a way to study detailed physical and
chemical properties that allows for scrutiny of fundamental principles and mechanisms and may
lead to technological and methodological developments.
It has recently been shown by several groups4
that it is feasible to make measurements in a
junction containing exactly one molecule. Such studies constitute the logical place for the science
of molecular electronics to begin.
1.2.1 Single Molecule Electrical Detection
Wiring individual molecules into an electronic circuit is an exciting idea that has been pursued
actively by many groups. To date, many molecules with wonderful electronic properties have
been identified and more with desired electronic properties are being synthesized in chemistry
labs5
.
Current work in molecular electronics usually addresses molecular junctions6, 7
and their
electronic transport properties8-10
. The conventional point of view (held in the classical Drude-
Sommerfeld or the quantum mechanical Kubo theories) is that conduction is the flow of current
in response to an electric field. An alternative point of view was put forward in 1957 by Rolf
Landauer (IBM, Yorktown Heights), who proposed that "conduction is transmission". This idea
is generalized to metal/single molecule/metal molecular nanojunction structures1
.
Although recent advances have been impressive, a basic question that remains a subject of debate
is what is the resistance of a simple molecule, covalently attached to two electrodes? Large
disparities have been found between different experiments11
, which reflect the difficulty of
forming identical molecular junctions. Even if the resistance of a molecular junction is
reproducibly measured, ensuring that the resistance is really due to a single molecule is another
substantial challenge.

8. 8
By bounding the junctions to electrical measurement devices, the molecules can be electrically
detected by making a change in the conductance. Since to date, there is no single molecule
conductance detected as reference, one could not declare that the detected current corresponds to
a specified number of molecules, more precise, on a single molecule. So the challenging subject
of electrical detection of a single molecule still is the subject of debate.
1.2.2 Single Molecule Optical Detection
In addition to electronic properties, many molecules possess rich optical, magnetic,
thermoelectric, electromechanical and molecular recognition properties, which may lead to new
devices that are not possible using conventional materials or approaches.
Different methods were used for single-molecule measurements which had their own limitations
due to the kind of the molecule and the instruments involved12
.
STM (scanning tunneling microscope) has played, and will continue to play, an important role in
the understanding of electron transport in single molecules because of its capability for both
microscopy and tunneling spectroscopy. As STM requires well-defined surfaces, such as single
crystals, it is not suitable for many devices or samples. AFM (atomic force microscopy) is
another useful structural characterization tool. Although it has lower resolution than STM, a
combined force and conductance measurement with AFM provides unique information about the
bonding nature of molecule-electrode contacts.
The most confidential way to be assured of the presence of a single molecule is to detect it by
optical means. Due to the special physics of this type of experiment, it is not possible to detect a
molecule trapped in a junction by simple microscopy techniques. It has to be discriminated from
its background environment.
Since the principal goal in this experiment is to “see” what is being measured, the optical
detection is essential. Among the detecting techniques, the fluorescence analysis is particularly
attractive since fluorophores could be excited and detected selectively. The fluorescence signal of
a single molecule, on the other hand, is independent of the dimensions of the detection volume
and remains constant. The fluorescence spectroscopy has an excellent sensitivity which is highly
required, because single molecule detection refers to single photon detection, but it’s the
“selectivity” that plays the key role in case of this experiment.

9. 9
1.2.3 Single Molecule Optical Detection Limits
There are also several limits in optical detection of molecules. To observe the fluorescence
emitted by a single molecule the optical back ground and the associated noise must be
sufficiently small. In a simple experiment, where a drop of the solution containing molecules is
located on a cover slip the most important criteria is to yield a low background scattered light and
good signal-to-background contrast.
In more complex systems, e.g. when the behavior of the molecules next to a metallic pattern over
the transparent media is studied, the discrimination of the sample has the priority importance
compared to signal/noise ratio.
Another limit in this type of detection is the photobleaching process. When molecules of interest
are photolabile (all molecules at room temperature), single molecule detection experiments are
limited by the total number of photons emitted before photobleaching.
1.3 Planned experiment
The main idea is to build up a compatible system which permits to apply both electrical and
optical detection techniques at the same time, so the optical detection will be an eyewitness for
the electrical measurement. This is a very challenging and novel idea. The goal of this work is to
present a strategy for constructing adequate electrodes patterning, suitable for optical
measurements, and to provide proof-of-principle indications that the molecules can be optically
revealed next to the electrical connectors.
1.3.1 Outlook
A device containing 8 junctions is designed and patterned with gold on coverslips with a precise
thickness of 170 mµ (Figure 5-2, Figure 5-3). These substrates are essential for being optically
compatible with optical microscopy techniques, with magnifying objectives precisely designed
for observations through such windows (Figure 7-4). The fluorescent probe in form of a solution
is passed over the junctions through the microfluidic channels.

10. 10
The solution is planned to be so diluted to reach the concentration for having only one molecule
per junction volume ( 3
100nm ). The ensemble is connected through the gold reference electrodes
to highly sensitive electrical measurement devices which indicate the changes in the conductance
and resistance of the junction. The whole system is placed under a microscope with a laser beam
and a camera together which permits to excite the molecules while passing over the junctions
(Figure 2-2). The laser beam and the camera are focused on the junction so once a molecule
approaches the junction area it can be detected optically and if a detected single molecule trapped
in the junction comes along with a peak in the conductance curve, it could be considered as a
single molecule’s conductance.
2 Total Internal Reflection Fluorescence Microscopy (TIRFM)13
TIRF is so named because it involves the total internal reflection of fluorescence excitation light.
For total internal reflection to occur, several criteria must be met. When light passes from a
medium of higher refractive index to a medium of lower refractive index, the light is refracted
away from the normal at the boundary. At higher angles of incidence there reaches a point where
the light will not transmit into the lower refractive index medium and will instead be totally
reflected. This angle is known as the critical angle, and its value depends on the refractive indices
of the media (n1, n3):
1 1
3
sinc
n
n
θ −  
=  
 
This phenomenon is known as Total Internal Reflection. This condition is achieved in the
microscope with a laser illuminating from the periphery of the back focal plane of an objective
lens. The immersion oil (1.515) and coverslip (1.52) are the higher refractive index media while
the aqueous media (1.36 for Ethanol) is of a lower refractive index. Even though the light is
totally reflected according to traditional optical theory, a small amount of energy does pass
through this interface into the lower refractive index media in the form of an evanescent wave.
"Evanescent" means "tends to vanish", which is appropriate because the intensity of evanescent
waves decays exponentially with the distance from the interface at which they are formed. If this
energy is not absorbed, it passes back into the glass.

11. 11
However, if a fluorophore molecule is within the evanescent wave it can absorb photons and be
excited (Figure 2-1). In this way, it is possible to get fluorescence with a very low background of
excitation light. The penetration depth for a TIRFM evanescent wave is 50-100nm (100nm in this
experiment). Only the fluorescence molecules within the shallow penetration of the evanescent
wave will be visible, allowing very selective, high contrast fluorescence imaging12, 14
.
Figure 2-1 - Schema of the excited fluorophores with the evanescent wave, (left: cross section view of the glass
slide, right: setup for TIRF mictoscopy)
Figure 2-2 – Inverted microscope configuration for TIRFM

12. 12
3 Fluorescent probe
There are several restrictions that limit the choice of fluorescent probe, as listed below:
• The Argon laser sources used to excite the fluorophores has a wavelength equal to 488nm.
The adequate fluorescent probe must have a maximum excitation wavelength of 488nm.
• The fluorophore needs to have a chemical group (cyanate, thiol, etc...) in order to bind to
gold edges.
• It is important that the chosen probe be a commercial product and available in stock for
further needs.
The first choice was Ruthenium Bipyridine complex because of its compatible excitation
wavelength and being well known as a fluorescent dye. The idea was to bind a thiol group at one
of the ends so the molecule could bind to metal which came out to be a complex chemical
process with a low probability of succeeding. The next best choice was the fluorescein dye15
.
Fluorescein dye is one of the most common fluorescent probes, investigations on its spectral
properties indicate that it can exist in several ionization forms (cation, neutral, anion and
dianion), each of which has distinct spectral properties16
.
Figure shows only the prevalent forms in aqueous solution. At various pH values fluorescein
equilibrates between several ionization states without having a characteristic peak of a
predominate form.
Figure 3-1 – A four-state molecular switch (A=absorbance, λ =wavelength)

13. 13
3.1 Fluorescein Isothiocyanate
Fluorescein isothiocyanate (FITC) is a yellow-green colored low molecular weight. FITC is
excitable at 488nm, close to its absorption maximum at 494nm, and produces maximum
fluorescence emission around 520nm. It is commonly conjugated with antibodies for use in
indirect immunofluorescence15, 17
, and it’s a commercial product.
FITC is the original fluorescein molecule functionalized with an isothiocyanate group (-N=C=S),
replacing a hydrogen atom on the bottom ring of the structure.
Systematic Name Fluorescein isothiocyanate
Chemical Formula 21 11 5C H NO S
Appearance Orange – Yellow powder
Molecular Mass 389.4 gr/mol
Excitation maxλ =495 nm
Emission maxλ =525 nm
Table 1, Fluorescein Isothiocyanate properties
FITC is tested for solubility and solution appearance at 1 mg/ml in acetone to give a clear yellow
solution. It is soluble in anhydrous dimethyl sulfoxide (DMSO) at 5 mg/ml.10 It is soluble in
water at less than 0.1 mg/ml in water, at 20 mg/ml in ethanol and at 9 mg/ml in 2-
methoxyethanol.1 An organic solvent for stock solution is advised, since FITC decomposes in
water. The product is light-sensitive, and should be stored dry and in the dark at 2 °C to 8 °C.
Since there are several materials in direct contact with the solution, the choice of solvent becomes
limited. The best compatible solvent which is at the same time organic and does not react with
the medium is Ethanol.
The ideal concentration for having one molecule per junction volume would be 4
6.23 10 gr
lit
−
× .
The best way to reach this concentration would be the dilution.10mgr Of FITC powder was
solved in 20ml of ethanol. Two times 1mlit of the solution was diluted in 20ml of ethanol, and
the last time 9ml of the solution was diluted in 20ml of ethanol to reach the ideal concentration.

14. 14
The concerned calculation is given below:
3
1 3
1 1 2 2
3 3
2
2
2
3
3
3
4
4
10 ( ) 20 ( )
10 10
0.5
20 10
0.5 1 10 20 10
2.5 10
1.25 10
9
6.23 10
mg FITC ml Ethanol
grc
lit
c v c v
gr lit c lit
lit
grc
lit
grc
lit
v mlit
grc
lit
−
−
− −
−
−
−
→
×
= =
×
=
× × = × ×
= ×
= ×
=
= ×
The chemical structure, the absorption and fluorescence emission spectra and the fluorescence
emission spectrum of Fluorescein dissolved in ethanol are shown in Figure 3-2, Figure 3-3 and Figure
3-4.
Figure 3-2 - the chemical structure of Fluorescein, ethanol
Figure 3-3 - the absorption and fluorescence emission spectra

15. 15
Figure 3-4 - the fluorescence emission spectrum of Fluorescein dissolved in ethanol
Figure 3-5 - Fluorescein Isothiocyanate
3.2 Bleaching of FITC17, 18
Bleaching of FITC seems to involve at least to different processes occurring in parallel:
a) the rapid initial loss of fluorescence intensity during the first microseconds of excitation,
which appears to be reversible; and b) a more gradual bleaching process most probably caused by
irreversible photodecomposition of the dye and increasing with higher energies or longer times of
continuous excitation.
The first rapid and reversible process turned out to be highly sensitive to changes in the chemical
and physical microenvironment of the dye molecule. It is therefore of crucial importance to work
under strictly standardized conditions for this kind of experiment.

16. 16
4 Microfluidics:
4.1 Introduction
Both single-molecule detection (SMD) methods and miniaturization technologies have developed
very rapidly over the last ten years. By merging these two techniques, it may be possible to
achieve the optimal requirements for the analysis and manipulation of samples on a single
molecule scale. While miniaturized structures and channels provide the interface required to
handle small particles and molecules, SMD permits the discovery, localization, counting and
identification of compounds19
.
Embedded in microchips a few centimeters in size, tiny structures and channels are used to
efficiently separate, transport and analyze very small amounts of sample. Besides the reduced
sample consumption, the downscaling of reaction systems leads to a multitude of advantages over
macroscale systems.
By integrating different functional units for reaction, detection and separation into a microfluidic
channel network, it could become feasible to realize one microchip comprising all features of a
complete lab, which could be used to perform complex reactions and analyses.
Decreasing the dimensions of reaction systems to small volumes with low amounts of analytes
naturally stimulates the demand for adequate, high sensitivity detection techniques. Fluorescence
measurements are usually employed, as they are non-invasive and provide high temporal and
spatial resolution for a suitable experimental set-up.
4.2 Single Molecule detection in microfluidic channels19
The implementation of single-molecule techniques to microfluidic platforms offers additional
tools. Once the molecule is flowing through the detection volume, any photons emitted are
detected. To obtain a high signal, the excitation intensity could be set to almost saturation
conditions, since the dwell time of molecules is short in fast flowing streams and bleaching is a
minor issue.

17. 17
However, extremely high purities are required for the sample and the material used for the
microstructures in order to keep the background signal low. Capillaries or microchips of high
transparency are usually chosen (such as glass, or PDMS bonded on glass slides) that also enable
the simple positioning of the channel on a microscope stage.
4.3 Microfluidic devices
For widespread use of microfluidic systems in everyday tasks, simple devices that can easily be
integrated and interfaced with existing equipment and work flows are necessary. Microfluidic
capillary systems that autonomously flush sequences of nanoliters through a reaction chamber
fulfil the ease-of-usage requirements20, 21
.
4.3.1 PDMS (polydimethyl siloxane)
In microfluidic applications, polydimethyl siloxane (PDMS) is a popular type of polymer used in
the rapid prototyping of microfluidic systems. There are several advantages of using PDMS
including fast fabrication time; compatibility to a variety of biological and cellular applications
due to the high gas permeability of PDMS, ease of installation of fluidic interconnects from the
macroworld to the microfluidic device due to the elastomeric nature of PDMS, and so on.
In addition, a thin layer of PDMS can also be used as a capping layer for microfluidic devices as
it is optically transparent down to 280nm and thus suitable for a number of fluorescence or
absorbance detection schemes. PDMS is the most widely used silicon-based organic polymer,
and is particularly known for its unusual flow properties. It is optically clear, and is generally
considered to be inert, non-toxic and non-flammable. The chemical formula for PDMS is
(H3C)[SiO(CH3)2]nSi(CH3)3, where n is the number of repeating monomer [SiO(CH3)2] units.
4.3.2 Fabrication of microfluidic channels: Soft Lithography22
Soft Lithography is a microfabrication process in which a soft polymer is cast onto a mould that
contains a microfabricated relief or engraved pattern. Such devices allow nanoliters or picoliters
of aqueous liquid to be controlled and manipulated on-chip for a large number of applications.

18. 18
In the simplest case, the mould is made of a wafer substrate and a photopatterned SU-8 resist,
both of which are coated with a thin, fluorinated anti-adhesion layer. The mould containing the
relief of the microfluidic network are made through the photolithography process which entails
designing a transparency mask with the desired microfluidic network, spinning an appropriate
photoresist onto a silicon wafer, exposing the wafer to ultraviolet light, and developing the wafer
to produce the desired pattern. Next, the PDMS (in this experiment Sylgard 184 silicone
elastomer curing agent) is poured on the mould to form a layer about 3-4 mm thick. Then put in
the vacuum chamber to evacuate any entrapped air, by pumping air and vacuuming several times
with an increasing time table and at least in the oven in 75 C° for about one hour.
After, the polymer is peeled off the mold, and the surface of the polymer that was in contact with
the mold is left with an imprint of the mold topography. Such topography typically defines
channels and chambers that will form part of a microfluidic system.
Figure 4-1 – mould containing the pattern of the microfluidic network obtained by photolithography
Figure 4-2 – curing the PDMS over the patterned mould to form the channels, soft lithography

19. 19
5 Sample preparation:
5.1 Sputtering
Thin films are obtained by sputtering. The substrates are the coverslips with a precise thickness
of170 mµ . The aim is to sputter 15nm of Chromium as an adhesive layer (also could use
Titanium) and make two sets of samples by depositing Gold with the two different thicknesses of
60nm and 80nm.
Before placing the coverslips in sputtering chamber, their surface have to be cleaned, by putting
them in Acetone then Ethanol bath and let each one 5 minutes in ultrasound bath separately, then
dried and placed in the sample holder.
To obtain the deposition time to reach the determined thicknesses, the deposition rate is
calculated which is 3.75 Angstrom per seconds for Gold and 0.1 Angstrom per seconds for
Chromium. The deposition time will be 150 seconds for 15nm of Chromium, 213 seconds for
80nm of Gold and 160 seconds for 60nm of Gold.
18 coverslips are placed in the low temperature sample holder in sputtering chamber, deposited
60nm of Gold on 9 and 80nm of Gold on the rest.
To obtain very clean surfaces, an etching process through the plasma is programmed for 10
minutes after obtaining the desired vacuum pressure and before starting the deposition.
A second generation of the thin films used in this experiment is made by evaporation in Besancon
cleanroom facility.
5.2 Microfabrication process in clean room environment
Optical lithography (photolithography) is used to engrave the designed pattern on the thin films
which consists of several steps, one after another with a precise time table. These processes are
performed in clean room environment to avoid at the highest level the impurities and in order to
have a pattern which matches the most its pre-designed mask.

20. 20
• Cleaning
First of all thin films have to be cleaned. First in Acetone and then in Ethanol bath separately,
each one left for 5 minutes in ultrasound bath, at the end dried with the nitrogen gun. Since the
thin films are on coverslip substrates, they are so fragile and would break easily. So it’s so
important to treat them carefully during the whole process.
• Spin Coating
The next step is called spin coating which is well known in thin film applications.
A typical process involves depositing a small puddle of a fluid photo resist (MICROPOSIT
S1813) onto the center of a substrate and then spinning the substrate at high speed (typically
around 3000 rpm). Higher viscosity and or larger substrates typically require a larger puddle to
ensure full coverage of the substrate during the high speed spin step.
Centripetal acceleration will cause the resin to spread to, and eventually off, the edge of the
substrate leaving a thin film of resin on the surface. Once stopped spinning, left for several
seconds to dry.
Figure 5-1 - Spincoating
• Baking
The photo resist-coated wafer is then "soft-baked" or "pre-baked" to drive off excess solvent, at
100 °C for 2 minutes on the hot plate.
• Optical Lithography
After pre-baking, the photoresist (Shipleys’ Microposit S1813) is exposed to a pattern of intense
light. Optical lithography typically uses ultraviolet light (UV). The mask contains two series of
designs for both negative and positive lithography.

21. 21
Because of the positive photo resist, the positive window of the mask is used. After aligning the
sample and the mask, to the position where the minimum gap between them is reached,
ultraviolet light is being exposed.
Figure 5-2 – The design of positive mask containing the junctions
• Photoresist developing
Positive photoresist, the most common type, becomes less chemically robust when exposed,
negative photoresist becomes more robust. This chemical change allows some of the photoresist
to be removed by a special solution, called "developer" by analogy with photographic developer.
MF26A developer which is 2.38% tetramethyl ammonium hydroxide in water is being used as a
developer. Samples go through the etching process in developer for 30 seconds, after that for 20
seconds in distillated water and dried at the end.
• Checking filter
Before running the final etching program, the samples must be checked with the microscope. If
there are not successful ones among them, they must go back to the first step of the process.

22. 22
• Final Etching Process
Once good results and a clean pattern with the microscope are observed, the samples go through
the final etching process using KI/I2 (saturated KI (Potassium Iodid) solution + I2 (Iodine)) to
etch Gold and chromium etchant from Sigma Aldrich to etch chromium. Pre-backed for 90
seconds samples are put 30 seconds in KI/I2 then 15 seconds in distillated water then dried and
again 30 seconds in the acid and 15 seconds in distillated water and then dried. The pattern
becomes completely visible in form of gold lines on a transparent glass background.
Figure 5-3 – Gold patterns on coverslip
• Photolithography for windows of photoresist
The next process will be preparing the samples for deposition of a silicon oxide layer all over the
sample except on the junctions. So a typical way is to put the photoresist on the junctions so these
parts will stay uncovered after etching. The photolithography steps are repeated but with another
mask which is specially designed for this aim.
After checking the samples with the microscope and pre-baking for 90 seconds, the samples are
ready to go through the next steps of the experiment.

23. 23
5.3 Microanalysis and microfabrication
5.3.1 Focused Ion Beam (FIB)
FIB, resembles a scanning electron microscope (SEM). With the difference that iinstead of using
an electron probe, as in SEM, a Gallium ion probe is used in the FIB. Gallium is chosen because
it is easy to build a gallium liquid metal ion source (LMIS). In a Gallium LMIS, gallium metal is
placed in contact with a tungsten needle and heated. Gallium wets the tungsten, and a huge
electric field (greater than 108
volts per centimeter) causes ionization and field emission of the
gallium atoms. The ions are extracted and accelerated from the source through the column and
finally focused by electrostatic lenses onto the specimen. The fact that ions are several
magnitudes heavier than electrons will result in a much higher impulse when the ions hit the
surface of the specimen. This will lead to removal of the outermost part of the sample, so-called
milling. The FIB can be used for milling, imaging and also material deposition.
5.3.2 FEI Strata 235 Dual Beam
The Dualbeam is a combined FIB and SEM work station. Imaging can be performed with both
techniques along with FIB milling. This allows the DualBeam to "slice and view", where the
SEM automatically takes micrographs as the ion source mill. After image reconstruction a 3D
model can be reconstructed of the material microstructure. It also allows a small section of a
specimen to be cut and lifted out from the specimen using an Omniprobe micromanipulator. It is
also equipped with a gas injection system for platinum, tungsten and insulator deposition.
FEI Dual Beam 235 (FIB-SEM-STEM) with an ion beam (FIB, (5 nm) and electron beam (SEM,
(1 nm) which allows state-of-the-art nanofabrication of samples (characterization, deposition,
milling, etc.) is used to form the gap in the junction area by milling the thickness of gold and
substrate.
Carbon paste is also used to conduct the electrostatic charges to the ground to avoid charge
accumulation on the surface which leads to an unwanted shift of image and alignment errors.
Figure 7-3 shows the general form of the junction area before (a) and after (b) the FIB cut.
The image on the right gives a view from top, while the left one gives a cross section view.

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Figure 5-4 – before (a) and after (b) the FIB cut, right image: view from above, left image: cross section view
Figure 5-5 – SEM image of the junction with 79.5 nm gap obtained in FIB cut
6 Device preparation
6.1 Bonding between PDMS and passivated silicon23
The bonding quality of the PDMS capping layer is critical in microfluidic applications which in
general are designed to process very small amounts of fluids. Without a good seal, leakage of
fluids will inevitably occur due to a breakdown of the bonding between the two layers.
Oxygen plasma treatment has been used by many researchers to create an effective bonding of
PDMS to PDMS or glass. Plasmas can improve adhesiveness by removing surface contaminants,
introducing roughened bonding surfaces and reactive chemical groups.

25. 25
In particular, the –O-Si(CH3)2- unit in PDMS can be converted to silanol group (–OH), thus the
PDMS surface chemistry changes from hydrophobic to hydrophilic.
Once cured, the PDMS was peeled from the mould and divided into pieces using a razor blade for
bonding to the glass-gold samples.
The PDMS piece containing the microfluidic channels and the sample were first treated by
oxygen plasma and immediately brought in contact for bonding. The gas used for plasma
activation is oxygen. The plasma power used was set to 3.30 W while the plasma exposure was
maintained for 40 seconds. After placing the two pieces together the specimens were put on the
hotplate at 70 °C for 5 minutes with a weight placed on it to improve the bond strength.
Figure 6-1 – Sample with bonded PDMS and inserted tubes to guide the liquid
6.2 Wire Bonding
For electrical measurements, PCB chips are connected to the reference electrodes on the sample
via wire binding and via BNC connectors to the electrical devices.
6.3 Electroplating
Electroplating is the process of using electrical current to coat an electrically conductive object
with a relatively thin layer of metal. One application is to build up thickness on undersized parts.

26. 26
The deposition of a metallic coating onto an object is achieved by putting a negative charge on
the object to be coated and immersing it into a solution which contains a salt of the metal to be
deposited (in other words, the object to be plated is made the cathode of an electrolytic cell).
The metallic ions of the salt carry a positive charge and are thus attracted to the object. When
they reach the negatively charged object (that is to be electroplated), it provides electrons to
reduce the positively charged ions to metallic form. Figure 1 is a schematic presentation of an
electrolytic cell for electroplating a metal "M" from an aqueous (water) solution of metal salt
"MA".
Figure 6-2 - Electrolytic cell for electroplating
The gap made in FIB step which is about 100 nm large becomes less than 10nm by
electroplating.
7 Optical Measurements
Experiments are performed using a dedicated inverted microscope equipped for TIRF which
comprises an Olympus Microscpe using a PLAPON 60XOTIRFM Objective, a CCD Roper
Photonmax camera and a high power excitation by visible blue (488nm) from an Argon ion laser
source.

27. 27
A specific imaging method used in this experiment is based on FRET (Fluorescence Resonance
Energy Transfer) microscopy, and provides two simultaneous images at two different
wavelengths. The advantage is that one of the channels is tuned for TIRF measurements of the
specific fluorochrome used, and the other provides an image by transmission at higher
wavelength, therefore allowing us to simultaneously observe the sample pattern.
The reflected light is divided by two; each ray passes a filter and gives an image.
The image at right has a lowered background noise where the fluorescence is well obvious, and
the image on the left has the transmission mode properties where the background is clearer and
the fluorescence effect is less detectable compared to the image on the right.
By superposing the left and right images, the fluorescence and the strong background signal
would be observable at the same time which overcomes the problem of low back ground signal in
TIRFM.
7.1 Detection of fluorescent Beads
Fluorescence of beads with diameter equal to 1 micrometer was observed as a test experiment for
TIRF.
Figure 7-1 – Fluorescent beads (diameter =1 mµ ) detected by TIRF

28. 28
7.2 Fluorescent Probes
A drop of the solution containing FITC is placed on the coverslip. The concentration of the
solution will be the same in all of the steps of the experiment. ( 2
2 2.5 10c −
= × ) which is 300 times
bigger than the ideal concentration for a single molecule.
In the TIRF mode, and the low background signal, the bright points representing a single
fluorophore or a group of fluorophores can be easily detected. The fast bleaching of FITC is
observed which is shown in Figure 7-2 and Figure 7-3. (The total time for recording the
bleaching process was 500 microseconds)
Figure 7-2 – Bleaching process of FITC obtained by TIRFM
Figure 7-3 – Bleaching process of FITC obtained by TIRFM, very low background signal

29. 29
Figure 7-4 – left: PLAPON 60XTIRFM objective, right: sample holder in TIRFM setup
Figure 7-5 – Sample placed in a drilled Petri dish to be compatible with the TIRFM sample holder
7.3 Final observation
A drop of the solution is put on the sample with gold lines. The fluorophores are detected near to
gold edges. Figure 7-6 represents the image obtained by transmission, the black lines are gold and
the bright areas represent the coverslip. The visible window on the junction is put in the FIB cut
step which is about 2 mµ wide and 10 mµ long.
Figure 7-7 and Figure 7-8 show the TIRF measurements of the same setup. The bright points
represent one or a group of fluorochromes. These images show that it is indeed possible to obtain
a fluorescence image, next to a transmission image giving a clear insight into the sample position.

30. 30
Figure 7-6 – transmission image of fluorophores over the coverslip containing gold patterns
Figure 7-7 TIRF detection

31. 31
Figure 7-8 TIRF detection
8 Results and discussion
The fact of observing the fluorescence next to Gold patterns is so close to the principal goal of
the experiment. There are several experiments pointing out the change of fluorescence next to
gold24-26
which can be either enhancing or quenching. It is possible that the known behavior of
fluorochromes changes completely in an environment similar to that of this experiment.
One of the most prominent problems in this type of experiments is the rapid bleaching of
fluorescent intensity of FITC which occurs within fractions of milliseconds of illumination and
which is in direct dependence to the time energy× product of excitation. Thus, for the study of
this fluorescence kinetics it turned out to be essential to use very fast shutters, photometers and
recording devices17, 27
.

32. 32
Another suggestion would be using fluorescent microspheres and beads instead of fluorochromes.
These structures are less complex and it is easier to make them compatible with the favorite
conditions.
TIRFM is the technique used for several years to detect single molecules and gaining popularity.
Fluorescence detection with two photon excitation could be a technique in parallel with TIRFM
which has its own advantages and disadvantages.
In optical measurements with the presence of microfluidic channels, no fluorescence was
detected. There are several arguments that may be able to explain this phenomenon.
• The influence of the microenvironment of the FITC molecule on its bleaching and
fluorescence properties17
.
• The possibility of sticking the FITC molecules inside the PDMS tunnel.
• The amount of liquid flowing in the channel is not enough for fluorescence detection of
TIRFM.
• The special physics of the system, its fragility and location above the microscopes’
objective made so many unwanted movements and loosing the focus.
• Because of the limited area for moving the objective, it was not possible to move between
channels and junctions. Thus, repeating the experiment with different samples would be
so important, which could not fit in the fixed time frame for this experiment.

33. 33
9 Conclusion
A structure containing gold junctions with microfluidic channels over them was fabricated on a
glass coverslip. This setup represents a compatible system with the optical microscopy
inspection. This success opens the way to observe the sample by optical techniques when
performing electrical measurements, which go beyond TIRFM experiments.
Images obtained in TIRFM measurements point out the accomplishment that, in principle, it is
possible to use this particular method for single molecule detection on this type of samples.

34. 34
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