Thesis Lysanne Mol 29 06 2020

Summary
In tumor cells, hypermethylation occurs, which means that promotors from tumor suppressor genes are methy-
lated. This results in the genes being silenced, which enables increased proliferation in tumor cells. Since the
specific hypermethylated DNA (hmDNA) pattern differs for each cancer type, hmDNA is a very promising
biomarker for cancer. hmDNA from various cancer types can be found in urine, which enables a non-invasive
isolation method.
The BIOS research group at the University of Twente is working on a microfluidic device to enable early
detection of cancer from urine. The process consists of several steps, among which an optical detection step
that uses plasmon resonance. Plasmon resonance is the result of the interaction between the quasi-free con-
ducting electrons of a metal nanoparticle and an incoming electromagnetic wave. The effects can be seen in
the near-field as a locally enhanced field and in the far-field as scattering spectra. When a metal nanoparticle
approaches a metal film, plasmonic coupling takes place, which enhances the plasmon resonance effects. In a
scattering spectrum, this can be seen as a red-shift. This effect is used in the optical detection step of the
microfluidic device: a gold nanoparticle (AuNP) is connected to a gold film via a folded DNA hairpin. When
the target DNA binds, the hairpin unfolds, and the distance between the AuNP and the film increases. This
can be seen as a blue-shift in a scattering spectrum, which can be measured by a dark-field microscope.
One of the questions that is asked with this detection method is how one can distinguish different positive
signals from each other when multiple DNA sequences are detected simultaneously (multiplex sensing). A
possible answer to this question might be to look at the movement of the AuNP over time. This movement
influences the scattering signal over time, since this spectrum depends on the distance between the particle and
the film. Therefore, the goal of this research is to evaluate various influencing effects on plasmon resonance and
the study the possibility of multiplex testing using scattering fluctuation over time.
This research is split up into two parts. First, the influence of gap size, AuNP size and the incident angle
of the incoming light wave on the near-field and far-field effects is studied. For this, the MNPBEM (Metal
Nanoparticle Boundary Element Method) toolbox in MATLAB is used. The results were used to find the op-
timal set-up for the second part of the project. Here, the MNPBEM simulations were combined with a model
made by Laurens Spoelstra which describes the AuNP movement over time. The possibility of multiplex testing
was studied for different particle sizes, tether lengths, camera frame rates and filters.
It was found that according to the simulations, multiplex testing is possible using 80 nm AuNPs and DNA
tethers that differ only 10 basepairs in length. This can be done by measuring the dynamic variance in red-
green ratio. It is recommended to continue testing with different tether lengths and AuNP sizes within the size
standard deviation range to fully evaluate this resolution. The simulations could be improved by including the
microscope detection efficiency, filter imperfections and quantum tunneling and electron nonlocality effects. It
was also advised to validate the different simulated results using experimental data. Altogether, this research
is a promising step towards fast, early-stage cancer detection on a microfluidic device.
Samenvatting
In tumorcellen vindt hypermethylatie plaats, wat betekent dat de promotors van de tumorsuppressorgenen
gemethyleerd worden. Hierdoor wordt de expressie van deze genen onderdrukt, wat zorgt voor versterkte
proliferatie in tumorcellen. hmDNA is een veelbelovende biomarker voor kanker, gezien het specifieke hyper-
gemethyleerd DNA (hmDNA) patroon verschilt per kankertype. hmDNA van verschillende kankertypen wordt
gevonden in urine, waardoor DNA op een niet-invasieve manier te isoleren is.
De BIOS onderzoeksgroep van de Universiteit Twente is een microfluidisch systeem aan het ontwikkelen om
kanker in een vroeg stadium te kunnen detecteren uit urine. Het systeem bestaat uit verschillende stappen,
waaronder een optische detectiestap dat gebruik maakt van plasmon resonance. Plasmon resonance is het
gevolg van de interactie tussen semi-vrije electronen in een metaal nanodeeltje en een elektromagnetische golf.
De effecten zijn zichtbaar in het nabije veld als een lokaal versterkt electrisch veld en in in het verre veld als een
scattering spectrum. Wanneer een metaal nanodeeltje een metaaloppervlak nadert, vindt plasmonic coupling
plaats, waarbij de plasmon resonance effecten versterkt worden. In een scattering spectrum is dit te zien als
een roodverschuiving. Dit effect wordt gebruikt in de optische detectiestap in het microfluidische systeem: een
goud nanodeeltje (AuNP) is verbonden met een goudoppervlak via een gevouwen DNA hairpin. Wanneer het
tumor DNA bindt, opent de hairpin, waardoor de afstand tussen het gouddeeltje en het oppervlak toeneemt.
Dit is te zien als een blauwverschuiving in het scattering spectrum, wat gemeten kan worden met behulp van
donkerveldmicroscopie.
Een van de vragen die gesteld worden bij deze detectiemethode is in hoeverre het mogelijk is om onderscheid
te kunnen maken tussen de verschillende positieve signalen wanneer meerdere DNA sequenties tegelijkertijd
gedetecteerd worden (multiplex-detectie). Een mogelijk antwoord op deze vraag is om te kijken naar de beweg-
ing van een gouddeeltje over de tijd. Deze beweging beı̈nvloedt het scattering signaal over de tijd, gezien het
spectrum afhankelijk is van de afstand tussen het deeltje en het oppervlak. Daarom is het doel van dit onder-
zoek om de verschillende factoren die plasmon resonance beı̈nvloeden te onderzoeken en om te onderzoeken of
multiplex-detectie mogelijk is met behulp van scattering fluctuatie over de tijd.
Dit onderzoek is opgedeeld in twee delen. Eerst wordt de invloed van de afstand tussen het deeltje en het
oppervlak, de grootte van het deeltje en de invalshoek van het licht op de effecten in het nabije en verre veld
bestudeerd. Hiervoor wordt de MNPBEM (Metal Nanoparticle Boundary Element Method ) toolbox in MAT-
LAB gebruikt. De resultaten worden gebruikt om de optimale opstelling voor het tweede deel van het project te
bepalen. Hier worden de MNPBEM simulaties gecombineerd met een model gemaakt door Laurens Spoelstra
waarin de beweging van het gouddeeltje over de tijd wordt beschreven. De mogelijkheid tot multiplex-detectie
is onderzocht voor verschillende deeltjesgroottes, DNA ketenlengtes, camera frame rates en filters.
In de simulaties is vastgesteld dat multiplex-detectie mogelijk is wanneer gebruik gemaakt wordt van 80 nm
gouddeeltjes verbonden aan DNA keten die slechts 10 basenparen in lengte verschillen. Dit wordt gedaan door te
kijken naar de dynamische variatie in rood-groen ratio. Er is aanbevolen om verder te testen met verschillende
ketenlengtes en deeltjesgroottes binnen het bereik van de grootte standaard deviatie om deze resolutie verder
te evalueren. De simulaties kunnen verbeterd worden door microscoopdetectie efficiëntie, filter imperfecties,
kwantum tunneling en elektron non-lokaliteit mee te nemen. Er wordt ook geadviseerd om de simulaties te
valideren met behulp van experimentele gegevens. Al met al is dit onderzoek een veelbelovende stap richting
snelle detectie van kanker in een vroeg stadium met behulp van een microfluidisch systeem.
Contents
1 Introduction and Literature Overview 1

1.1 DNA hypermethylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Microfluidic device for hmDNA detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.3 Research goals and expectations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Theory 4
2.1 Theory behind plasmon resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.1.1 Localized surface plasmons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.1.2 Excitation of localized surface plasmons . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.1.3 Damping of the oscillating effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.1.4 Dipole approximation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.1.5 Near-field and far-field effects of plasmon resonance . . . . . . . . . . . . . . . . . . . . 6
2.1.6 Plasmon coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2 Current research on plasmon resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.3 Conclusion on the theory behind plasmon resonance . . . . . . . . . . . . . . . . . . . . . . . . 10
3 Influencing Factors of Surface Plasmon Resonance: Script Development 12

3.1 Specifications of the simulated system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.2 Script development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.3 Script validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.3.1 Validation using simulated data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.3.2 Validation using experimental data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.4 Effect of different layers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.5 Conclusions on script development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
4 Influencing Factors of Surface Plasmon Resonance: Simulations 18

4.1 Effect of incident angle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
4.2 Effect of AuNP size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
4.3 Effect of gap size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
4.4 Conclusions on the influencing factors of surface plasmon resonance . . . . . . . . . . . . . . . . 24
5 Scattering Fluctuation of a Particle-on-Film System over Time: Script Development 25

5.1 Look-up table for varying gap sizes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
5.2 Simulated camera settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
5.2.1 Measuring time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
5.2.2 Filter influence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
5.3 Script development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
5.4 Conclusions on script development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
6 Scattering Fluctuation of a Particle-on-Film System over Time: Simulations 28

6.1 Time-dependent scattering intensity for different tether lengths . . . . . . . . . . . . . . . . . . 28
6.1.1 Simulating 40nm AuNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
6.1.2 Simulating 80nm AuNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
6.1.3 Relation between tether length and mean scattering intensity . . . . . . . . . . . . . . . 29
6.2 Influence of standard deviation of particle size . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
6.3 Red-green ratio for different tether lengths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
6.4 Dynamic variance of RG-ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
6.5 Conclusions on scattering fluctuation over time . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
7 Discussion and Recommendations 35
7.1 Discussion on simulated results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
7.2 Suggestions for further experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
7.2.1 Imaging techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
7.2.2 Experiments to validate the effect of influencing factors . . . . . . . . . . . . . . . . . . 36
7.2.3 Experiments to validate the scattering fluctuation over time . . . . . . . . . . . . . . . . 37
7.3 Suggestions on further simulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
7.3.1 Further simulations on influencing factors . . . . . . . . . . . . . . . . . . . . . . . . . . 37
7.3.2 Further simulations on scattering fluctuation over time . . . . . . . . . . . . . . . . . . . 37
8 Conclusion 39
Bibliography 40
Appendices 42
CHAPTER 1. INTRODUCTION AND LITERATURE OVERVIEW
Chapter 1
Introduction and Literature Overview
1.1 DNA hypermethylation

DNA methylation is one of the epigenetic modifications of DNA and is responsible for regulating the gene
expression. When DNA is methylated, a methyl group is bound to the genome, usually to a cytosine within a
CG dinucleotide. Methylating the promotor of a certain gene silences gene expression by restricting the access
of the transcriptional machinery, which makes DNA-methylation one of the ways in which gene expression is
regulated [1]. CG nucleotides are often concentrated in CpG islands, which are around 1-2 kilo base pairs (kb)
long [1–3]. These islands usually contain promotors. However, in tumor cells, hypermethylation occurs, which
means that methylation occurs more frequently than in normal (healthy) tissue [1]. This way, tumor-suppressor
genes can be silenced, which is one of the causes of increased proliferation in tumor cells [1, 4]. Hypermethylated
DNA (hmDNA) is found in many cancer types, making it a possible biomarker to distinguish cancer cells from
cells from normal tissue [1, 2, 5, 6].
DNA from cancer cells can be found in the blood as circulating tumor DNA (ctDNA) due to extracellular vesicles
containing DNA from tumor cells, or due to apoptotic or necrotic tumor cells [6]. Small DNA fragments can
pass the kidneys and end up in urine [7, 8]. This results in the possibility to extract hmDNA from urine in
a non-invasive way. This DNA can then be analyzed to test for various cancer types in an early stage [6].
Previous studies on hmDNA as an indicator for cancer have shown however that multiple DNA sequences
should be analyzed to obtain a high sensitivity [6, 9].
1.2 Microfluidic device for hmDNA detection

The BIOS research group at the University of Twente uses this knowledge on hmDNA to create a device to
enable early detection of cancer from urine [3]. This device would be a microfluidic device, which would make
point-of-care testing possible. The process in this chip consists of several steps, as can be seen in Figure 1.1.
The first three steps consist of isolation, enzymatic restriction and amplification of the hmDNA, to provide a
DNA sample with a sufficient concentration for the detection. The last step is the optical detection, which is
the focus of this research.
The optical hmDNA detection step is shown schematically in Figure 1.2. The detection device consists of a gold
surface on which gold nanoparticles (AuNPs) are immobilized via DNA hairpins (DNA capable of binding to
itself). These hairpins have a sequence complementary to the target DNA. Due to the small distance between
the AuNP and the gold film, electromagnetic coupling between the AuNP and the gold surface can take place,
which results in a red-shift of the scattering spectra (this process will be described more elaborately in Section
2.1.6). When the target DNA is present in the urine sample, it will bind to the complementary DNA of the
hairpin, which results in the unfolding of the hairpin. As a result, the distance between the nanoparticle and
the surface increases, which is visible as a blue-shift of the scattering spectrum. This optical change can be
observed using a dark-field microscope. By creating different regions with different sequences of hairpin DNA,
various cancer indicating DNA sequences can be tested. Since the hypermethylation of DNA is sequence specific
for each tumor type, the origin of the cancer cells can be found.
However, as explained before, multiple DNA sequences are needed to indicate the origin of the tumor with a
high sensitivity. Therefore, it is desired to test multiple DNA sequences on the same chip while still being able
to distinguish the positive signals from each other, a process called multiplexed sensing. One way multiplexed
sensing can be enabled is be creating DNA hairpins of different lengths. The AuNPs will move in a certain
pattern over time, depending on the length of the DNA tether and the size of the AuNP. This movement can be
seen as fluctuations in the scattering signal, since the scattering depends on the distance between the particle
and the film (which will be described in more detail later on).
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Figure 1.1: A schematic overview of the different steps in detecting hmDNA on chip. The focus of this
research will be on the last step, the optical detection of the hmDNA on chip (J.E. van Dongen, MSc., personal
communications, 29-04-2020).
Figure 1.2: A schematic overview of the sensing of DNA using gold nanoparticles (AuNPs) connected to a gold
surface via DNA hairpins (J.E. van Dongen, MSc., personal communications, 29-04-2020).
1.3 Research goals and expectations

The goal of this research is to study the optical response of an AuNP on a gold film for different AuNP sizes
and gap sizes to determine the best AuNP size and DNA tether length difference for multiplex sensing. This
overall goal is split up into two parts. During the first part, the effect of different gap sizes, AuNP sizes and
incident angles on the optical response will be evaluated. At the end of this part, a choice will be made for the
optimal incident angle and AuNP size(s) for the next part.
The formulated research question is ”What is the effect of gap size, AuNP size and incident angle on the
scattering cross sections and near-field plots of AuNPs on a gold film?” Several hypotheses were formulated,
based on the theory described in the next chapter:
• It is expected that with increasing gap size, the intensity of the near-field enhanced field will be reduced
and the scattering plots will show a decrease in scattering intensity as well as a blue-shift.
• It is expected that the effects of increasing gap size are most significant for changes in the lowest gap
sizes, such that the effect of changing gap size becomes less with a more and more increasing gap size.
• It is expected that with increasing particle size, the near-field enhanced intensity will increase and the
scattering plots will show an increase in scattering intensity and a red-shift. It is also expected that the
spectrum will show broader peaks as a result of increased dampening.
• It is expected that for increasing incident angle, the intensity of the longitudinal dipole resonance peak
will increase and the intensity of the transversal dipole resonance peak will decrease.
During the second part of this research, the scattering spectra will be combined with a model which will be
made by Laurens Spoelstra. His model describes the AuNP position over time. By combining the models it
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becomes possible to predict the fluctuations in scattering wavelength over time. It is expected that different
DNA tether lengths have characteristic fluctuations in their scattering signal over time. The goal of this is to
determine the length difference needed between two different dsDNA tethers in order to be able to distinguish
them from each other based on the fluctuation in scattering signal over time. Together with Laurens, the
following hypotheses were formulated, based on theory which is mostly described in Laurens’s thesis [10]:
• Longer dsDNA tethers show more variation in z-height (gap size) and thereby show a larger variation in
plasmon shift.
• In the case AuNPs are connected to longer, but different lengths of dsDNA, they may both lose plasmon
coupling. This might make it impossible to distinguish due to low scattering intensity.
• Larger beads show more scattering at larger gap sizes (due to the fact that larger beads result in higher
scattering intensity) and they diffuse slower, giving rise to a slower change in position.
First, the theory behind plasmon resonance will be described. Then, the thesis splits up into two parts, just
as the research. The first part is called ”Influencing Factors of Surface Plasmon Resonance” and answers the
first research question. Therefore, Chapter 3 and 4 describe the script development and simulations done, after
which the first question is answered. The second part is called ”Scattering Fluctuation of a Particle-on-Film
System over Time” and is the result of a cooperation with Laurens Spoelstra. Here, it is tried to reach the
second goal of this research, which is described in Chapter 5 and 6. In Chapter 7, all the results are discussed
and some recommendations are done for future research, for experiments as well as simulations. In the last
Chapter, all the conclusions from this thesis are summed up.
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CHAPTER 2. THEORY
Chapter 2
Theory
In this chapter, the theory needed to achieve the research goal is explained. For this, the theory behind plasmon
resonance (including plasmon coupling) is discussed and the visualisation and evaluation of these effects are
described.
2.1 Theory behind plasmon resonance

2.1.1 Localized surface plasmons
Conducting materials such as metals have quasi-free conducting electrons, which are not bound to a single atom
and can move freely [11, 12]. These electrons are capable of sustaining electromagnetic charge density waves
(plasma waves), which are known as plasmons [11, 13, 14]. There are two types of plasmons: bulk plasmons and
surface plasmons. Surface plasmons are bound to the interface between the plasma (the metal) and a dielectric
medium [11, 13, 14]. In nanoscale particles, the surface plasmons are confined to a smaller space, which is why
they are called localized surface plasmons (LSPs) [15, 16].
2.1.2 Excitation of localized surface plasmons

When metal nanoparticles (MNPs) are exposed to an electromagnetic field, the electrons get displaced from
their normal equilibrium position, creating separate charges at both sides of the nanoparticle. However, opposite
charges will exert a restoring force to shift the electrons’ positions back to equilibrium. Therefore, the shifted
electrons and the restoring force act as an oscillator, moving with a certain resonance frequency. This effect is
called plasmon resonance [11, 17]. The motion of this oscillating system is described by [17]:
e
x00 + γx0 = E0 e−iωt (2.1)
me
Where x is the displacement, γ is the damping term due to scattering, e is the electron charge, me is the
electron mass, E0 is the amplitude of the external electric field and ω is the frequency of the external electric
field. As can be seen in Equation 2.1, the behavior of the oscillator model is influenced by the mass, charge
and density of the electrons and the geometry of the particle [11].
2.1.3 Damping of the oscillating effect

The oscillating movement of the electron cloud surrounding the MNPs is dampened by several interactions of
the electrons [11, 17]:
• Bulk damping: the result of the absorption of light in the metal. For gold, this is significant for wavelengths
shorter than 600 nm.
• Radiation damping: the result of the absorption and emission of photons by the electrons. This is linearly
proportional to the amount of oscillating electrons and thereby to the size of the particles.
• Electron-surface scattering: the result of electrons scattering on the surface of the particle, losing energy
in the process. This is significant for gold particles smaller than 40 nm, since this is the electron mean
free path in gold. The electron mean free path is the average distance an electron travels between two
interactions. Therefore, in particles smaller than the electron free mean path, the scattering at the surface
will be significant, since very few interactions will take place when travelling from one side of the particle
to the other.
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CHAPTER 2. THEORY
• Electron-defect scattering: the result of energy loss after electrons scatter on defects in the metal lattice.
These factors all contribute to the damping effect, which is visible in the broadening of the scattering linewidth.
2.1.4 Dipole approximation

The response of a material to the electric field is described by the dielectric constant and is the consequence of
the interaction of the electrons in the material to this field [18]. It is related to the refractive index, since the
refractive index is the square root of the dielectric constant. In general, the dielectric constant is a function of
the angular frequency ω and has a real and imaginary part.
In case the nanoparticles are much smaller than the wavelength of the light that is used to excite the plasmons,
the dipole approximation is valid. This implies that the electric field within the particle is approximately
homogeneous and that the electrons in the nanoparticle will move in phase [11, 17]. The dipole moment (µ) of
a single electron within the nanoparticle can now be given by [17, 18]:
µ = ex (2.2)
The macroscopic polarization, meaning the polarization of the entire nanoparticle, can now be calculated by
multiplying the dipole moment of a single electron with the electron density in the nanoparticle:
P = ne ex (2.3)
The dielectric function can in general be calculated by:

0 E 0 + P ne ex
(ω) = =1+ (2.4)
0 E 0 0 E0
Where e0 is the vacuum permittivity. When Equations 2.1 and 2.4 are combined, the Drude dielectric function
of gold can be found [17, 18]:
ωp2
(ω) = 1 − (2.5)
ω(ω + iγ)
Where the plasma frequency is given by: s
ne e2
ωp = (2.6)
me 0
As can be seen in Equation 2.6, the plasma frequency only depends on the electron density, which means that
it only depends on the material. For simplicity, the contributions of the bound electrons in the material are
ignored.
The dielectric constants of the environment and the particle together with the particle size influence the plasmon
resonance effect [11]. The polarizability of the metal nanoparticle is given by the Clausius-Mossotti relation
[11, 17]:
m (ω) − d
m (ω) = 3V (2.7)
m (ω) + 2d
where m and d are the dielectric constants of the metal and the surrounding dielectric respectively and V is
the volume of the nanoparticle. This polarization reaches a maximum on the frequency at which:
m (ω) + 2d = 0 (2.8)
Which gives an approximation for the localized surface plasmon resonance resonance (LSPR) frequency when
combined with Equation 2.5 and when assuming the damping term to be zero (γ = 0):
s
ωp2
ωLSP ≈ (2.9)
2d + 1
This LSPR frequency can be observed as a maximum in the scattering intensity after interaction with light at
this specific frequency, as will be discussed later.
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CHAPTER 2. THEORY
2.1.5 Near-field and far-field effects of plasmon resonance

As described in the previous sections, the electrons around the nanoparticles oscillate in phase with the external
electromagnetic field due to the balancing of the force of the electromagnetic field with the restoring force. This
effect can be seen in Figure 2.1A. This movement of the electron cloud results in a locally enhanced electro-
magnetic field [11, 17, 19], which is simulated and visualized in Figure 2.1B. This increased electromagnetic
field is the near-field effect of plasmon resonance, but also indirectly induces a far-field effect. As can be seen
in Figure 2.1C, the electromagnetic field of the nanoparticle and the external field of the incoming light wave
overlap, resulting in constructive and destructive interference [11, 17, 19]. In the first case, this leads to scat-
tering in all directions (Figure 2.1D). In the latter case, the light gets absorbed, which can also be measured.
The effects of scattering, absorption and extinction together form the far-field effects.
Figure 2.1: A schematic representation of the effects of plasmon resonance. (A) The oscillation of metal
nanoparticles and their electron clouds with respect to the external electric field. (B) Simulated increased
electric field around a metal nanoparticle. (C) A metal nanoparticle with its localized electric field (red) and
the external electric field (blue). (D) A graphical representation of a simulated scattering response as a function
of the wavelength [17].
Since the electric field around the particle is larger than the particle itself, the scattering, absorption and
extinction cross sections will always be larger than the nanoparticle itself. The size of these cross sections
can be calculated using Mie theory. Mie theory is based on Maxwell’s equations and describes the scattering
cross-section (Csca ), extinction cross-section (Cext ) and absorption cros-section (Cabs ) [11, 18]:
∞
2π X
Csca = 2 (2n + 1)(|an |2 + |bn |2 ) (2.10)
K n=1
∞
2π X
Cext = (2n + 1)Re(an + bn ) (2.11)
K 2 n=1
Cabs = Cext − Csca (2.12)

Where an and bn describe the decomposition of the electromagnetic field and k is the wave vector in the
surrounding of the nanoparticles. k can be found using the frequency of the external electric field, the dielectric
constant of the nanoparticle and the speed of light [18]:
p
ω (ω)
k= (2.13)
c
Lysanne Mol 6
CHAPTER 2. THEORY
In case of the dipole approximation, a and b can be defined as:
i2(1 − m /d )y 3
a1 ≈ (2.14)
3(2 + em /ed )
b≈0
The distinction between near-field and far-field can be found when looking at the equations which describe the
electric fields generated in radial and polar directions [17]:
cos(θ) 2 n 2 d
Er = ( + )|µ(t − nr/c)| (2.15)
4π0 r3 c r2 dt
sin(θ) 1 n 1 n2 1 d
Eθ = ( 3+ + )|µ(t − nr/c)| (2.16)
4π0 r c r2 c2 r dt
In the near-field domain, which is at r < λ, the term with r−3 dominates. The far-field domain is set at r > 2λ,
where the term r−1 dominates. In between lies the intermediate domain, where r−2 is the strongest. In other
words: the near-field effect are very strong, but decline very fast when one moves away from the nanoparticle.
After that, the far-field effects dominate.
2.1.6 Plasmon coupling

When two metal nanoparticles move close to each other, the electromagnetic fields
of both particles become coupled, which is called plasmonic coupling [11, 13, 17].
There are two different ways in which this coupling can take place, namely per-
pendicular or parallel (see Figure 2.2). In case of parallel polarization, the net
charge is reduced, since the nanoparticles can ’feel’ the presence of the opposite
charges of the other particle. As as result, a red-shift in resonance frequency
can be seen in the far-field. The dipole field in between the particles is stronger,
since the electromagnetic fields overlap, resulting in an increase in local field en-
hancement [11, 13]. In case of perpendicular polarization, the two dipole fields
enhance each other, which increases the restoring force. In the far-field, this can
be seen as a blue-shift in the resonance frequency [11, 17, 20]. In the near-field,
destructive interference between the electromagnetic fields will take place, result-
ing in a doughnut-shaped scattering pattern, which can be observed in dark-field
microscopy images [13].
Which polarization takes place depends on the polarization of the light. In case of
p-polarized light, the electric field is parallel to the planewave, which means it has
components in the horizontal and vertical direction. Thereby, it can excite both
the transversal (destructive coupling) and the longitudinal dipole (constructive
coupling). The electric field of s-polarized light on the other hand is perpendic-
ular to the planewave. Since this field only has a horizontal component, it can Figure 2.2: The two differ-
only excite the transversal dipole. The different coupling modes can be seen as ent coupling states, perpen-
different peaks in the scattering plots (see Figure 2.3). In this case, the reso- dicular (destructive) and
nance wavelength of the transversal dipole is always lower than the resonance parallel (constructive), and
wavelength of the longitudinal dipole. In case of asymmetry of the system, the their effects on the reso-
quadrupole dipole mode occurs, which has the lowest resonance wavelength of all nance frequency(ωρ ). Im-
three [17, 21]. In case of a quadrupole, half of the electron cloud moves parallel age adapted from [11].
to the applied electric field, while the other half moves anti-parallel [21].
The effects of plasmon coupling can be used in several different ways, some examples will be given later.
Lysanne Mol 7
CHAPTER 2. THEORY
Figure 2.3: The different coupling modes can be seen in different resonance peaks in the scattering plot: (1)
Quadrupole dipole, (2) Transversal dipole, (3) Longitudinal dipole [17].
Plasmon coupling is also observed for film-coupled nanoparticles, in which nanospheres are connected to a
metal film via a tether or separated by a thin spacer layer. In this case, the nanoparticle couples with the
delocalized surface plasmon polariton in the film. In other words: the particle will act as if there was another
nanoparticle on the other side of the film (see Figure 2.4). The main influences in the plasmon resonance
with film-coupled nanoparticles are the particle size and shape, the incident angle of excitation, the distance
between the nanoparticle and the film and the composition of the nanoparticle and the film [13, 17]. During
this research, the influence of all of these effects will be studied. However, the focus of this study will be on
the influence of the distance between the nanoparticle and the film (the ’gap’) and the particle size.
Figure 2.4: Schematic image of film-coupled nanoparticles. The nanoparticles couple to the surface as if
they are coupled to another nanoparticle on the other side of the film. Just as with interparticle coupling,
parallel/destructive coupling (figure A) and perpendicular/constructive (figure B) is seen [16].
When the gap size between the nanoparticle and the film increases, the intensity of the scattering decreases
and a blue shift takes place in the parallel coupling (see Figure 2.5) [13, 17, 20]. The reason for this is that the
coupling becomes less strong when the gap size increases, therefore the netto charge increases and with that,
the restoring force will also increase. This can be seen as an increased resonance frequency, in other words: a
blue-shift. In a research similar to this one, Van Liempt altered the gap size between an 45 nm gold film and a
100 nm AuNP from 1 to 10 nm, during which a 200 nm blue-shift took place [17]. For perpendicular coupling,
a red-shift will take place when the gap size increases, and the destructive interference will decrease. In the
near-field, the larger gap size will result in less overlap between both electromagnetic fields and thereby less
enhancement [17, 22]. Therefore, the intensity of the electromagnetic field will lower. This also results in a
lower amplitude in scattering effects. In the same research from Van Liempt [17], decreasing the spacing from
10 to 2 nm lead to an intensity increase of 450%. It is worth noting that most of these effects took place at the
Lysanne Mol 8
CHAPTER 2. THEORY
smallest spacing distances: after decreasing the spacing from 10 to 5 nm, only a 30% increase in intensity was
measured.
Figure 2.5: Left: Graphical representation of the effect of different gap sizes on the scattering plots (simulated).
As can be seen, the resonance wavelength and intensity decreases for larger gap sizes. Right: A graphical
representation of the effect of different nanoparticle sizes on the scattering plots (simulated). As can be seen,
the resonance wavelength and intensity increases with increasing particle size. Images adapted from [17].
When the particle size increases, the opposite charges in the particle are further apart. Therefore, the restoring
force decreases. This effect can be seen as a red-shift in the scattering spectra, since a larger restoring force
results in an lower resonance frequency [17, 23] (see Figure 2.5). An increase in particle size also results in more
oscillating electrons contributing to the field enhancement, which gives a stronger localized field enhancement
in the near-field. In the far-field, this can be observed as a higher intensity in the scattering spectra. As
mentioned in Section 2.1.3, an increase in particle size will also result in more radiation damping, which will
result in broader peaks in the scattering spectra.
There are three possible techniques to image the far-field effects of plasmon resonance (Figure 2.6A-C) [24].
Bright field microscopy is the most basic microscopy technique. With this technique, the sample is directly
illuminated by the light source, and the light falls directly on the objective. When objects absorb or scatter
some of the light, they become visible as a darker spot on the image (see Figure 2.6A). Advantages of using
this techniques are high illumination strength and easy alignment of the light beam. Drawbacks include that
objects with sizes in micrometer scale or smaller usually do not absorb or scatter enough light to appear as
fully dark spots in the image. This way, the contrast is not optimal. An additional problem are the diffraction
rings that appear, resulting in the fact that less particles can be observed simultaneously [24].
A second technique is dark field microscopy. Now, the beam of light is partially blocked in the center, such
that the light hits the sample at a large angle [17, 24]. The scattered light will be captured in the centre on the
objective. A detailed image of this system can be seen in Figure 2.6D. The scattering sample will show up as a
bright spot on a dark background (Figure 2.6B). While it is more difficult to get the alignment of the light beam
correct, the signal-to-background ratio is substantially increased compared to bright field microscopy [24].
Lastly, there is evanescent wave microscopy, which measures the change in evanescent waves within the pene-
tration depth of the light from a Total Internal Reflection Microscopy (TIRM) (Figure 2.6C). This way, only
the particles of interest, namely the particles close to the surface, are measured. However, with this technique
it is difficult to obtain a high light intensity, since the evanescent wave decays exponentially [24]. Therefore,
only particles directly attached or very close to the surface can be measured with sufficient intensity.
In comparing these techniques, it can be seen that dark field microscopy is the most appropriate one to image
the scattering effects of gold nanoparticles, which is why this technique is most used in researches on plasmon
resonance (e.g. [13, 20, 24, 25]).
Lysanne Mol 9
CHAPTER 2. THEORY
Figure 2.6: A-C: A schematic set up and microscopy image of three different imaging techniques ((A) bright field,
(B) dark field and (C) evanescent wave microscopy) to visualize the plasmon resonance effects of gold nanopar-
ticles. All microscope images are taken at a typical camera view with 90x magnification [24].D: Schematic
set-up of dark field microscopy. The light beam from the halogen light source is partially blocked by a central
aperture, thus only scattered light from the sample reaches the objective [17].
2.2 Current research on plasmon resonance

Over the last few years, many studies have been performed on plasmon resonance, of which a few will be
discussed. One of the researches that is comparable to the first part of this project (the part on influencing
factors of plasmon resonance) is described in an article from Armstrong et al. [22]. Here, a particle-on-film
system is used to evaluate the near-field and far-field effects of changing the thickness of the metal film and
the gap size. The system that is used is very similar to the system in this research, therefore the data from
Armstrong was used to validate the scripts (see Section 3.3). In Armstrong’s article, simulated data is compared
to experimental data, where the heterogeneity of the experimental data is also shown. Another research that
uses a particle-on-film system is that of Mock et al. [16]. Here, the influence of gap size is evaluated by
measuring the scattered light using dark field microscopy and TIR illumination. The data from Mock et al.
show the blue-shift for increasing gap size and also the doughnut scattering profile due to destructive coupling.
This study only showed experimental data, without any additional simulations. A study from Zhang et al.
[26] on the other hand uses both simulated and experimental data to evaluate near-field and far-field effects of
a particle-on-film system. Here, the influence of light polarizations on the plasmonic modes and thereby the
effect of incident angle are described.
A research that compares the best to the second part of this project is from Visser et al. [20]. Visser uses a
system of two metal nanoparticles which are connected through a tether molecule. When the conformation of
the tether changes, the distance between the two nanoparticles varies and a shift in plasmon resonance is seen.
Visser wants to use this system to evaluate the folding of e.g. polypeptides, DNA hairpins and certain proteins.
In his article, he models the position of the particles using Brownian dynamics, which he uses as an input in
the calculations of the scattering spectra to obtain scattering data over time. This is the same method used
in this project. The difference however is that Visser uses this method to evaluate the folding behavior of the
tether, while the goal of this project is to use the tether length dependence of the particle motion to create a
system in which two signals can be distinguished from each other by a small difference in tether length.
2.3 Conclusion on the theory behind plasmon resonance

To summarize this section briefly: when an electromagnetic wave hits a metal nanoparticle, the unbound con-
ductive electrons will move through the nanoparticle. This effect is counteracted by a restoring force, which
results in an oscillating electron cloud which moves in anti-phase with the external electric field. The degree
to which this effect takes place depends on the size of the nanoparticle and the dielectric constants of the
Lysanne Mol 10
CHAPTER 2. THEORY
environment and the nanoparticle. This last factor can be calculated using the Drude function. Using these
factors, the optical cross sections and the LSPR frequency can be calculated. These belong the far-field effects,
significant at a distance r > 2λ from the nanoparticle, while the near-field effects are the enhancement of the
local electric field at a distance of r < λ from the nanoparticle. When two nanoparticles approach each other,
plasmonic coupling will take place, which results in a red-shift in the resonance frequency and an even more
increased localized electromagnetic field. This effect becomes less with an increased gap size. An increase
in nanoparticle size will result in an increase in field enhancement in the near-field and red-shift, increased
intensity and broader peaks in the spectra from the far-field. These effects are best imaged using dark field
microscopy. Due to promising advantages over similar techniques, plasmon resonance is of great interest in
different fields. This is why much research has been done over the last few years, of which a few studies have
been described in more detail.
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CHAPTER 3. INFLUENCING FACTORS OF SURFACE PLASMON RESONANCE: SCRIPT
DEVELOPMENT
Chapter 3
Influencing Factors of Surface Plasmon

Resonance: Script Development
In this chapter, the specifications of the simulated system will be described. After this, it will be explained how
the script is developed using the MNPBEM toolbox in MATLAB, by describing the way the script is constructed
and the different choices that have been made. This script is validated using simulated results and experimental
results.
3.1 Specifications of the simulated system

The system consists of a sensor with a layer structure on which AuNPs are immobilized via DNA tethers.
The sensor in the experiments is a SPR sensor from Ssens, and the gold nanoparticles are ordered from BBI
Solutions. The layer structure of the sensor is constructed as followed: the bottom layer is 1 mm thick and
made of borosilicate glass with a refractive index of 1.51. The refractive index is important for the simulations,
since the dielectric constant is equal to the square of the refractive index. The second layer is a thin titanium
layer of approximately 5 nm thick, which ensures better adherence of the top layer of the sensor, namely the
gold layer. This gold layer is 50 nm thick. On top of the sensor, a self assembled monolayer (SAM) of undecane
is placed, to which PEG linkers are connected. These PEG linkers are connected to 25, 50 or 100 bp long DNA
tethers, onto which the gold nanoparticles (AuNPs) are connected. The nanoparticles themselves are 20, 40,
60 or 80 nm in diameter. The influence of the DNA tethers on the plasmon resonance is ignored. The SAM
and PEG layers are modelled, where the refractive index of the SAM layer is 1.405 [27] and the refractive index
of PEG is 1.37. The environment consists of 0.10 mM PBS, which has an refractive index of 1.338 [28]. A
schematic overview of the system can be seen in Figure 3.1
Figure 3.1: A schematic overview of the simulated system. The position of the different layers depends on the
particle radius (R) and the gap size (G). The dielectric functions of gold and titanium are taken from [29].
Lysanne Mol 12
DEVELOPMENT
3.2 Script development

The base of calculating the effect of plasmon resonance of a system are Maxwell’s equations. The MNPBEM
package for MATLAB evaluates these equations for metal nanoparticles (MNP) using the boundary element
method (BEM) [19]. BEM is a method in which only the boundaries between the different environments are
evaluated instead of evaluating the entire object. This leads to less unknowns, faster calculations and the possi-
bility to work with infinite domains [30]. For this, it is assumed that the dielectric environment is homogeneous
and isotropic and that the different dielectric functions are separated by abrupt interfaces [19].
There are two different ways in which the MNPBEM package can calculate the plasmon response after excita-
tion by an external EM field: using the quasistatic solver or the full BEM solver. The first one is the fastest
option and is valid for structures significantly smaller than the wavelength of the incoming light. The full
solver on the other hand is slower due to the numerous matrix inversions, however the results are closer to
experimental data [19]. Therefore, it is recommended to use this method for the final simulations, while the
quasistatic solver can be used to get approximate results. The results of the BEM solutions of the Maxwell
equations can be evaluated by comparing them to the Mie theory (described in Section 2.1.5).
There are different steps needed to create field enhancement plots and scattering plots, as is summarized
in Figure 3.2. As can be seen, each step is accompanied with one or several functions that are part of the
MNPBEM package. Between the three different simulations, only the incident angle, the particle size and the
location of the particle with respect to the gold film will be varied, which are part of step 3 (Create and locate
AuNP) and 4 (Define excitation wave and BEM options). The different steps will be explained briefly, together
with the choices made for these different steps:
• Define dielectric functions: in the class epstab, a table of dielectric functions is stored, which are either
constant (use epsconst) or stored in a table (use epstable). For the gold layer and nanoparticle and
the titanium layer, the dielectric functions from [29] are used. For other layers, the refractive index is
used to calculate the dielectric constants.
• Define layer structure: using the layerstructure class, the different layers in the structure are defined
in the correct order. This is only used for particles on a film. The layer structure is defined as described
before.
• Create and locate AuNP: using trisphere, the size and number of vertices are defined. Using shift,
the particle is placed on the right height above the film. The comparticle class combines the shape of
the particle with the dielectric functions. The number of vertices was set at 200 nm, since this corresponded
with the data used for the validation (see Section 3.3). The gap size is defined as the distance between
the PEG layer and the nanoparticle and was varied between 1 and 40 nm.
• Define excitation wave and BEM options: using planewave, an excitation wave from the set direction
is generated. In bemoptions, several choices can be made for the BEM solver, such as quasistatic
versus retarded simulation, adding a layer structure and activating a waitbar. The incident angle of the
excitation wave was varied between 10 and 70 degrees during the first simulations. After this, the angle
was set to 60 degrees, as motivated in Section 4.1. All simulations are done for unpolarized light, since
this matches the experimental set-up. The scattering was calculated for 100 wavelengths linearly spaced
between 450 and 1000 nm. For the near-field simulations, the wavelength was set at the peak(s) in the
corresponding far-field scattering plot. The BEM options were set such that a retarded simulation was
done while considering the influence of the layer structure.
• Precompute Green functions: using tabspace, a grid will be created, which will be the basis of the table.
Next, compgreentablayer is used to create the Green function table. Since this is a time-consuming
step, it is recommended to check if the tabulated Green functions already exist when executing the code.
However, since the Green functions depend on the particle size and position, for most simulations the
functions needed to be recalculated for every iteration.
• Solve equations using BEM: bemsolver solves Maxwell’s equations using BEM.
• Generate emission pattern: only for the near-field plot. In spectrum the direction of the incoming
light is defined. In farfield this is combined with the results from the bemsolver to compute the
electromagnetic fields.
• Compute electric field: only for the near-field plot. The meshfield object is first made by specifying the
particle, the positions and the choices in several options. In this object, the results from emesh will be
stored. emesh uses the result from the bemsolver as an input to calculate the induced electromagnetic
fields.
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DEVELOPMENT
Figure 3.2: Flowchart describing the different steps that need to be taken to evaluate the effects of surface
plasmon resonance. For each step, the different MNPBEM-specific functions used are specified. The first steps
are equal for both the field enhancement plot and the scattering plot. However, after the equations are solved
using BEM, the emission patterns and electric fields need to be calculate to create the field enhancement plot,
while the scattering plot can be created directly after solving the equations. The figures in the last step are
created using demo scripts from the MNPBEM-package.
3.3 Script validation

3.3.1 Validation using simulated data
First, the near-field and far-field plots were validated using simulated data from Armstrong [22]. The simulated
system consists of four layers: a glass substrate, a 5 nm chromium layer, a gold film and a polymer spacer
layer. On top of this, a 100 nm gold nanoparticle is placed in an environment of air. First, the validation of
the far-field scattering plots was done, as can be seen in Figure 3.3. The script for this validation can be found
in Appendix A. In this case, the particle was placed 5 nm above the gold film. It can be seen that the general
shape of the graphs, the locations of the peaks and the heights of the peaks are very similar.
The near-field electric fields were also validated (see Figure 3.4). For this, data from the master thesis [17] on
which the article from Armstrong is based was used, since the near-field plots were not used in the publication.
The script is given in Appendix B. It can be seen that the field enhancement is in the same order of magnitude
as in the simulations from Van Liempt. This is the most important aspect to validate, since the actual image
will most often not be used in the results. The differences in the way the images are created are personal pref-
erence and are therefore of very little importance. Therefore, it was concluded that this script was also accurate.
Lysanne Mol 14
DEVELOPMENT
Figure 3.3: Validation of the far-field scattering plot using data from the supplementary information from
Armstrong [22]. Figure A shows the simulated results and figure B shows the results from Armstrong. It can
be seen that the general shape and the location of the peaks are similar.
Figure 3.4: A) Simulated near-field electric field. B) Simulated data from Van Liempt [17]. The order of
magnitude for both field enhancements are roughly the same, and both figures show a field enhancement that
is localized in the gap between the particle and the film.
3.3.2 Validation using experimental data

The far-field plots are also validated using experimental data, since these plots are the most used in this research
and are therefore the most important. This validation is done using data from Mock et al. [16]. This system
consists of a 60 nm gold nanoparticle on top of a layerstructure with a glass layer, a 5 nm chromium layer, a 45
nm gold film and a polyelectrolyte spacer layer. The thickness of the spacer layer is differed to create different
gap sizes between the gold film and the gold particle. The script used in the previous validation has been
altered, as described in Appendix C. The results are shown in Figure 3.5. As can be seen, the location of the
peaks is roughly the same. The only difference is seen in the peak with the largest gap size. In the simulations,
this peak is split into two different peaks. This difference might be due to a different light polarization, since
the polarization of the light determines which dipole occurs (see Section 2.1.6).
Lysanne Mol 15
DEVELOPMENT
Figure 3.5: Validation of the far-field scattering plots using experimental data from Mock et al. [16]. Figure
A shows the simulated results, figure B shows the results from Mock. The resonance frequencies of the three
smallest gap sizes match those of Mock. The resonance peak of the largest gap size matches the general
resonance frequency, however, the peak is split in two, probably due to a difference in light polarization.
3.4 Effect of different layers

Now that the scripts are validated, the system described in Section 3.1 can be implemented. For this, first a
”base system” with a gold AuNP above a layerstructure consisting of a glass layer and gold layer was created.
Then, a titanium, SAM and PEG layer is added. To see the effect of these layers, the scattering is evaluated
after each layer is added, as can be seen in Figure 3.6. The simulations show that for every layer, the scattering
cross section lowers. Adding the titanium layer has little influence. This was to be expected, since the gold
layer functions as a near-perfect mirror, which lets very little light through [22]. Therefore, the titanium layer,
which is located beneath the gold layer, has little influence on the plasmon resonance. The SAM layer, which
is placed on top of the gold layer, has more influence, since it weakens the coupling between the particle and
the gold layer. Lastly, the PEG layer has less influence than the SAM layer, since it is thinner and its dielectric
constant is closer to that of the environment.
Figure 3.6: The influence of adding a titanium, SAM and PEG layer on the far-field plot. By adding each layer,
the scattering cross section decreases. The SAM layer has the largest influence. The influence of the titanium
layer is small, since it is placed below the gold layer. The influence of the PEG layer is smaller compared to
the SAM layer, since it is thinner and has a lower dielectric constant.
Lysanne Mol 16
DEVELOPMENT
Figure 3.7: The influence of adding a titanium, SAM and PEG layer on the near-field plot. Please note the
differences in the colorbars in Figure A-D. First, the near-field plots of A) the original system (glass layer, gold
layer, gold AuNP, PBS environment) B) the system when a titanium layer is added C) the system when a
titanium layer and a SAM layer are added D) the system when a titanium layer, SAM layer and PEG layer
are added. E) shows the maximum field enhancement for systems A-D. It can be seen that the titanium layer
barely has any influence on the field enhancement and that the field enhancement decreases when the SAM
and PEG layers are added.
The effect of adding the different layers has also been evaluated in the near-field, as shown in Figure 3.7. It can
be seen that adding the titanium layer has no influence on the near-field plot and maximum field enhancement.
This again proves that the gold layer acts as a near-perfect mirror, thereby blocking the influence of the titanium
layer placed below it. When the SAM and PEG layer are placed on top of the gold layer, the field enhancement
decreases, due to decreased coupling. The influence of the SAM layer is more significant than that of the PEG
layer, since the SAM layer is thicker and has a higher dielectric constant compared to the PEG layer.
3.5 Conclusions on script development

A script has been developed to evaluate the near-field and far-field effects of the incident angle of the incoming
light, the gap size and the AuNP size. The near-field and far-field plots have been validated using data from
Armstrong [22] and Mock [16]. After this, the system as imaged in Figure 3.1 is simulated, where the effect of
the different layers on the plasmon resonance was shown.
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CHAPTER 4. INFLUENCING FACTORS OF SURFACE PLASMON RESONANCE: SIMULATIONS
Chapter 4
Influencing Factors of Surface Plasmon

Resonance: Simulations
In this chapter, the effect of three different influencing factors will be evaluated using MATLAB simulations:
the effect of incident angle, gap size and nanoparticle size. The influence will be evaluated in the near-field
and the far-field. For incident angle and nanoparticle size, the results of the simulations will be used to chose
an incident angle and specific nanoparticle sizes for the simulations that will be done in the second part of this
research.
4.1 Effect of incident angle

One of the factors that influence the plasmon resonance effects, is the incidence angle of the incoming light.
The scattering effect was evaluated for two different AuNP sizes, 40 and 80 nm, and for four different incident
angles, 10, 30, 50 and 70 degrees. These sizes were chosen since the other simulations will also be done using
40 and 80 nm AuNPs, as will be explained in Section 4.2. For these simulations, a gap size of 2 nm was chosen.
The script that was used can be found in Appendix D. The results are shown in Figure 4.1 and Figure 4.2. For
40 nm AuNPs, it can be seen that all the angles result in a (large) resonance peak around 600 nm (peak (1)).
This peak is the result of the transversal dipole. The largest scattering cross section is obtained at an incident
angle of 50 degrees. At an angle of 30 degrees, the quadrupole dipole peak (peak (3)) is also visible. When the
incidence angle is only 10 degrees, the longitudinal peak (peak (1)) is barely visible.
When looking at the scattering plots of 80 nm AuNPs, it can be seen that the longitudinal dipole peak (peak
(1)) is far more visible than for 40 nm AuNPs. An angle of 70 degrees now results in the highest scattering
intensity for the longitudinal dipole peak, however, the difference with an incident angle of 50 degrees is min-
imal. The longitudinal dipole resonance peak now lies around 830 nm, while this was around 780 nm for 40
nm AuNPs. The transversal dipole (peak (2)) and quadrupole dipole peaks (peak (3)) are the lowest for the
incident angle of 70 degrees. At the other wavelengths, the intensities of these peaks are substantially higher,
and for incident angles of 10 and 30 degrees, these peaks are higher than the longitudinal dipole peak. At an
incident angle of 10 degrees, the longitudinal dipole resonance peak is not visible at all.
This behavior can be explained when looking back at the two different coupling modes, parallel (longitudinal
dipole) and perpendicular (transversal dipole) coupling. The angle of the incoming light and thereby the x-
,y- and z-components of the electric field determine which of the coupling modes occur. In case the incident
angle is 0, the electric fields of both p-polarized and s-polarized light only have components in the x- and y-
direction. Therefore, at very low incident angles, only the resonance peak from the transversal dipole is visible.
When the incident angle increases, the x-component and z-component of the electric field increases, while the
y-component remains the same. This can be seen as an increase in the longitudinal dipole and a decrease in
the transversal dipole [17].
Lysanne Mol 18
Figure 4.1: Scattering plots for 40 nm AuNPs located 2 nm above the gold surface. The incoming light had
an incident angle of 10, 30, 50 or 70 degrees. The longitudinal dipole peak (peak (1)), transversal dipole peak
(peak (2)) and quadrupole dipole (peak (3)) are visible in different degrees, depending on the incident angle.
The highest scattering intensity is obtained at an incident angle of 50 degrees.
Figure 4.2: Scattering plots for 80 nm AuNPs located 2 nm from the gold surface. The incoming light had
an incident angle of 10, 30, 50 or 70 degrees. The longitudinal dipole peak (peak (1)), transversal dipole peak
(peak (2)) and quadrupole dipole (peak (3)) are visible in different degrees, depending on the incident angle.
The highest scattering intensity for the longitudinal dipole peak is obtained at an incident angle of 70 degrees,
while the transversal dipole peak is higher for the other angles.
Lysanne Mol 19
The change in near-field effects as a result of a

changing incident angle was also evaluated, as
displayed in Figure 4.3 (for the corresponding
script, see Appendix E). It can be seen that the
local field enhancement increases for an increas-
ing incident angle. This is in agreement with the
theory described before, since an increasing in-
cident angle results in an increase in the x- and
z-component of the electric field. For both par-
ticles, an increase in incident angle above 50 de-
grees does not result in further increase in field
enhancement. This is most likely the result of
the decreasing transversal dipole for larger inci-
Figure 4.3: The simulated local field enhancement of 40 and
dent angles.
80 nm AuNPs at different incident angles. The nanoparticles
It can therefore be concluded that the scattering
are located 2 nm from the gold film. An increase in incident
is best measured using an incident angle of 50
angle results in an increase in field enhancement.
or 70 degrees. The dark-field microscope that
is used in the experiments that will follow these
simulations has Olympus MPLFLN-BD objectives, which have a numerical aperture (N.A.) of 0.8 and 0.9 at
50x and 100x magnification respectively [31]. This corresponds to an incident angle varying from 57 to 63
degrees (when the medium is assumed to be water). Therefore, in future simulations, an incident angle of 60
degrees is assumed, since this matches both the capabilities of the set-up and the results from the simulations.
4.2 Effect of AuNP size

The second influencing factor that has been researched is the size of the gold nanoparticles. Four different sizes
were tested: 20, 40, 60 and 80 nm. One script was used to evaluate both AuNP size and gap size, as seen
in Appendix F. The results are shown in Figure 4.4. It can be seen that the scattering intensity increases for
increasing nanoparticle size, as was expected. For 80 nm particles, the resonance peaks from the transversal
dipole and quadrupole dipole have also increased significantly with respect to the signal from 60 nm particles.
The longitudinal dipole resonance peak is also increasingly broad, which is the result of increased damping, as
explained in Section 2.1.3. Lastly, a slight red-shift is visible for increasing nanoparticle size. This is due to
the fact that for larger particles, the opposite charges are further apart, which results in a lower restoring force
(See Section 2.1.6). This leads to a lower resonance frequency, which can be seen in a red-shift.
Figure 4.4: Scattering plots for different sizes AuNPs located 2 nm from the surface. The scattering intensity
increases for larger nanoparticles. There is also a red-shift in resonance frequency visible.
Lysanne Mol 20
To make the relation between the AuNP size and the scattering cross section more clear, a range from 10 to 100
nm particles has been simulated. Figure 4.5 shows the total scattering cross section, which is equal to the area
under the scattering plot, as a function of the AuNP size. The simulated data has been fitted using the Curve
Fitting Toolbox in MATLAB. The formula of the fitted curve is f (x) = 0.0006664x4.845 with R2 = 0.9979.
When comparing this formula to the theoretical relation, some deviations are noted. According to literature,
the scattering cross-section of particles smaller than 100 nm is given by [32]:
128π 5 6 m2 − 1 2
σscat = R |( 2 )| (4.1)
3λ4 m +2
Here, m equals the ratio of refractive indices of the particle and the environment. One can see that both
the generated fit and Equation 4.1 are power functions. However, for the generated fit, the scattering cross
section increases with approximately R4.8 for increasing particle size, while this should be R6 according to
Equation 4.1. This can be explained by the fact that the equation is related to particles in an environment
with a single dielectric constant, while the simulated data point originate from a system of a particle connected
to a film. The interaction between the particle and the film might therefore change the relationship between
the AuNP size and the scattering cross section. This would also mean that the fitted curve should approach
Equation 4.1 for an increasing gap size, since the coupling between the film and the particle becomes less
with increasing distance. This theory is supported simulations that show that for a larger gap size, the power
increases: for a gap size of 10 nm, the formula of the fitted curve turns out to be f (x) = 3.584 ∗ 10−5 x5.454
(R2 = 0.9993). A final simulation with a gold sphere located in air without any film results in the fitted curve
f (x) = 1.825 ∗ 10−7 x6.078 (R2 = 1), which is in accordance with Equation 4.1 and thereby proves that the
simulations are correct.
Figure 4.5: Simulated scattering cross sections for different AuNP sizes, fitted by a curve with the formula
f (x) = 0.0006664x4.845 (R2 = 0.9979).
Figure 4.6: The local electric field for different AuNP sizes. It can be seen that the largest field enhancement
is concentrated in the gap in between the particle and the surface. Please note the difference in the colorbars.
The field enhancement increases for increasing particle size.
Lysanne Mol 21
Figure 4.6 shows the local electric fields for

different nanoparticle sizes. The maxima
of these fields can be seen in Figure 4.7.
Again, the results for the effect of AuNP
size and gap size were created with one
script, found in Appendix G. It can be seen
that increase in AuNP size results in an
increase in local field enhancement. The
graph also shows the same general shape
as Figure 4.5.
For the highest scattering intensity and
thereby the best signal for the dark-field
microscope, the largest particle size should
be chosen. However, considering that the
ultimate goal is to distinguish the bind-
ing of different DNA sequences by the Figure 4.7: The maximum field enhancement for different AuNP
movement of the particle, smaller parti- sizes. The field enhancement increases for increasing AuNP size.
cles could also be preferable, due to their The highest increase is found between 20 and 40 nm AuNPs.
larger diffusion coefficient. Therefore, it
has been decided to continue the simulations using two different AuNP sizes: 40 and 80 nm. In the second part
of this thesis, the optimal AuNP size will be determined based on the scattering-over-time simulations.
4.3 Effect of gap size

The last influencing factor is the effect of the distance between the particle and the film (the gap size). The gap
size was varied between 1 and 40 nm, since this was the expected gap range for the given DNA tether lengths
[10]. The resolution was set the highest for the lowest gap sizes (1 through 5 nm), since based on literature the
most change was expected here. The gap size was increased with 1 nm increments between 1 and 5 nm, with 2
nm between 5 and 7 nm, with 3 nm between 7 and 10 nm, with 5 nm between 10 and 20 nm and with 10 nm
between 20 and 40 nm. The effect of the gap size on the scattering cross section of 40 nm AuNPs can be seen
in Figure 4.8. As expected, the most change is seen between a gap size of 1 and 5 nm: The intensity of the
resonance peak around 750 nm decreases, while the resonance peak around 600 nm shifts to lower wavelengths.
For gap sizes larger than 6 nm, the scattering intensity in the red wavelength area decreases further and the
scattering intensity in the blue wavelength area keeps increasing, however, the resonance wavelength barely
shifts anymore.
Figure 4.8: The scattering intensity for 40 AuNPs at different wavelengths and AuNP sizes. A clear blue-shift
is visible for an increase in gap size.
Lysanne Mol 22
For 80 nm AuNPs, the blue-shift is more clear, since the peak around 750 nm has a higher intensity to begin
with, which makes the change in scattering wavelengths more prominent. It can also be seen that it takes
longer for the red-wavelength intensity to decrease than for 40 nm AuNPs (a change in gap size of 8 nm is
needed instead of 5 nm) (see Figure 4.9). This follows from the fact that larger AuNPs have stronger coupling
than smaller particles, therefore, a larger separation between the particle and the film is needed to significantly
decrease the coupling. The figure also shows that the overall linewidth is larger for larger particles, which was
also seen in Figure 4.4. Lastly, it can be concluded that the scattering intensity is much higher for 80 nm
AuNPs than for 40 nm AuNPs at all gap sizes and wavelengths.
Figure 4.9: The scattering intensity for 80 AuNPs at different wavelengths and AuNP sizes. A clear blue-shift
is visible for an increase in gap size.
The effect of a varying gap size has also been simulated in the near-field. Figure 4.10 shows the local field
enhancement for different gap sizes for both 40 and 80 nm AuNPs. The field enhancement is measured for the
wavelengths that correspond to the resonance frequency of the longitudinal dipole. For 40 nm AuNPs, it can
be seen that the largest decline occurs in the 1 to 5 nm gap size region, while this happens in the first 10 nm
for 80 nm AuNPs. This is due to the stronger coupling of larger particles. That is also why 80 nm AuNPs
show a higher local field enhancement for all gap sizes, as expected.
Figure 4.10: The maximum field enhancement of 40 and 80 nm AuNPs for different gap sizes. For an increasing
gap size, the local field enhancement decreases, with the strongest decline in the 1-5 nm region. The local field
enhancement of 80 nm AuNPs is overall higher than for 40 nm AuNPs.
Lysanne Mol 23
4.4 Conclusions on the influencing factors of surface plasmon reso-

nance
The effect of three different influencing factors has been evaluated: the effect of incident angle, AuNP size and
gap size. For the incident angle, it has been concluded that this factor determines which coupling mode(s)
are visible. This effect can be explained when one looks at the proportion of the x-,y-, and z-components in
the electric field of light for those different angles. It was concluded that the highest scattering intensity was
obtained for an incident angle between 50 and 70 degrees. Since the incident angle of the dark field microscope
that is used in the experiments is around 60 degrees, this angle was used in the rest of the simulations.
Next, it was concluded that an increase in AuNP size resulted in an increase in scattering intensity and a
red-shift of the resonance frequency, as was expected. Therefore, larger AuNPs will lead to better experimental
results. However, one should also keep in mind the second purpose of this research, namely the study of the
effects of the movement of bound AuNPs on the scattering signal. The ultimate goal is to be able to distinguish
two different signals from each other using this change in signal over time. Due to the diffusion coefficient of
80 nm AuNPs compared to 40 nm AuNPs, 40 nm AuNPs seem more suited for these studies. To balance these
two factors, further simulations are done using both 40 and 80 nm AuNPs.
Lastly, the effect of gap size was studied. It was found that with an increase in gap size, a blue-shift was visible.
This blue-shift took place in the 1-5 nm region for 40 nm AuNPs and in the 1-10 nm region for 80 nm AuNPs.
Lysanne Mol 24
CHAPTER 5. SCATTERING FLUCTUATION OF A PARTICLE-ON-FILM SYSTEM OVER TIME:
SCRIPT DEVELOPMENT
Chapter 5
Scattering Fluctuation of a
Particle-on-Film System over Time:
Script Development
Now that the effect of the different influencing factors is demonstrated, the model is combined with the model
made by Laurens Spoelstra which describes the AuNP motion over time [10]. This motion results in a variation
in gap size, inducing fluctuation in scattering intensity over time. In this chapter, the development of the script
for this second part of the project is described.
5.1 Look-up table for varying gap sizes

In the first part of this research, it was proven that the scattering plot is strongly dependent on the gap
size. Thus, when the position of a particle is not static but time-dependent, the scattering signal will also be
time-dependent. To convert the gap size over time to scattering intensity over time, it is needed to know the
scattering signal for each of these gap sizes. For this, a look-up table is created, which gives the scattering
signal for 100 wavelengths between 400 and 1000 nm for gap sizes between 0 and 40 nm. However, since it
would be very time-consuming and possibly redundant to evaluate every gap size between 0 and 40 nm with
0.001 nm precision, it was first evaluated what the resolution of the steps in gap size should be to obtain valid
results. The resolution was determined for different starting gap sizes. This is because it was shown in Section
4.3 that the scattering plots are very sensitive for a small change in gap size in the lower gap size range (lower
than 10 nm) and less sensitive for higher gap sizes. The sensitivity was evaluated for 1.5, 5, 7, 10, 15 and
20 nm gap sizes by increasing these gap sizes in different amounts and evaluating the change in scattering
intensity. Figure 5.1 shows the results for 40 nm AuNPs. The scattering intensity is determined by integrating
the scattering cross section plot within a wavelength range of 620 to 750 to obtain a measure of the intensity
of red scattered light. It must be noted however that in general, filters have a smaller range which will lead to
an overall lower intensity. The difference in sensitivity for different starting gap sizes can be seen and is most
clear for the larger steps (+0.4 nm and up). The same is done for 80 nm AuNPs, where the same trend was
found.
It was however still difficult to predict what change in scattering intensity would still be measurable. Therefore,
the step sizes were chosen such that the change in scattering intensity would be the same for all these steps.
With this, it has been decided to increase the gap size from 0 to 1 nm with 0.25 nm increments, from 1 to 5
nm with 0.5 nm increments, from 5 to 8 nm with 1 nm increments, and from 7 to 40 nm with 2 nm increments.
The look-up table was used in the script to find the closest gap size in the look-up table for each gap size in
the time series, to find the corresponding scattering signal.
Lysanne Mol 25
SCRIPT DEVELOPMENT
Figure 5.1: The simulated change in scattering intensity of red light for 40 nm AuNPs at different starting
gap sizes. Each time, the gap size is increased with a certain amount. The change in scattering intensity is
measured compared to the starting gap size, which is the reference point for the values on the x-axis.
5.2 Simulated camera settings

5.2.1 Measuring time
For correct simulations, it is important to know what the proper measuring time is. This measuring time
depends on the relaxation time (τ ), which is the time in which the tethered particle explores all possible
conformations [20]. This relaxation time was determined by Laurens as described in his thesis [10]. The
relaxation time turned out to be in the order of a few milliseconds. Thus, for a proper measurement, the
measuring time should be at least equal to a few milliseconds. However, it is recommended to measure at times
far longer than the relaxation time to ensure that all possible conformations occur multiple times. That is why
it was chosen to measure for 2 seconds in the simulations.
5.2.2 Filter influence

In case a high-speed camera is used, filters are needed to measure the intensity at specific wavelengths. In this
section, the optimal filter bandwidth is determined.
The reference points for each filter were the peaks in the red and green region in Figure 4.8 and Figure 4.9,
which are 760 nm and 600 nm respectively. From there, the filter was set for different bandwidths with the
reference point as centre wavelength. However, for the red wavelength region, the lower end of the filter would
go below 700 nm if this pattern was continued. This is undesirable, since then both the red peak and the green
peak would be captured with this filter. Therefore, for larger bandwidths, the filter was continued with a set
minimum of 700 nm while increasing the maximum passing wavelength for increasing bandwidth. For each
bandwidth, the difference in mean integrated scattering cross section between measurements with 50 and 60 bp
tethers was determined. The goal was to find the filter with the highest difference in mean scattering, since this
would contribute to the possibility of multiplex testing. The results can be seen in Table 5.1. A few interesting
things can be taken from this. First, it can be seen that the intensity in the red-wavelength region for 40 nm
AuNPs is very low such that it is overall not recommended to use this for the measurements, regardless of the
filter bandwidth. It can also be seen that in general, a higher filter bandwidth results in a higher intensity
and thereby a higher difference in mean scattering. This would advocate for using the largest filter bandwidth
possible. However, in the green-wavelength region, it can be seen that the largest filter bandwidth results in
a decrease in mean scattering compared to the second to largest filter bandwidth. This can be explained by
the fact that the range is larger than the peak one wants to measure, which results in the fact that also the
intensity of another peak is measured. This is not desirable, therefore, the bandwidth of the filter should be
chosen with this in consideration.
Lysanne Mol 26
SCRIPT DEVELOPMENT
Table 5.1: The influence of filter bandwidth on the variation in scattering intensity. The difference in mean
scattering intensity is calculated for 40 and 80 nm AuNPs connected to 50 bp and 60 bp long tethers. The
difference in scattering intensity is given in nm3 .
40 nm AuNP 80 nm AuNP
Filter bandwidth Red Green Red Green
40 4.3 0.0149e04 8.9632e04 4.4705e04
80 8.7 0.0117e04 1.4885e05 5.3413e04
120 18.1 0.0227e04 1.8104e05 5.2727e04
160 16.8 0.0308e04 1.9833e05 5.2762e04
200 15.5 0.0380e04 2.0700e05 5.0993e04
The filters that will eventually be used for the experiments will be custom made. For now, it is unclear what the
possibilities are for this and thereby what filter bandwidth can be achieved. Therefore, while it is recommended
to use a larger bandwidth, the simulations are continued using filters with 40 nm bandwidth. As a results,
the simulated data is a ”worst case scenario” with the lowest variation in mean scattering. The results can
be improved in simulations or experimentally by increasing the filter bandwidth. For now, it will at least be
proven what the minimal variation is that will be measured.
5.3 Script development

The final script for evaluating the scattering over time using data from Laurens’s model can be found in
Appendix H. The different sections of this script will now be explained. The sections on plotting the figures are
self explanatory and are therefore not mentioned here. Some sections are more applicable for Laurens’s thesis,
therefore more information will be given there.
• Settings: Here, certain settings are set. The thickness of the SAM layer is defined, since Laurens’s script
defines the gap size as the distance between the particle centre to the top of the gold layer and this needs
to be converted to the distance between the top of the SAM and the bottom of the nanoparticle. Besides
that, some settings are set for the fitting algorithm.
• Load the data files: The data containing the particle movement is loaded here. Then, for each selected
file, the important variables are read and saved to structs. In these structs, each row represents one file.
• Tau curve fitting: Here, the autocorrelations are calculated to find the constant τ for each file. τ deter-
mines the time over which the data should be averaged, as will be explained in Laurens’s data in more
detail [10]. The results from this section are not further discussed in this thesis, since they are more in
line with Laurens’s results.
• Camera averaging and filter settings: For the interpolation step, the data is resampled to make the
interpolation step faster. Besides that, the settings for the (simulated) camera and filter are set here.
These settings can be changed, depending on preference.
• Data processing: Here, all the calculations that are needed for the conversion from gap size over time to
scattering over time are done. First, the gap sizes are calculated, since the gap size is defined differently in
Laurens’s model [10]. Then, resampling takes place with the settings set at the previous section. All the
data is interpolated to determine the gap sizes in the look-up table that are the closest to the gap sizes in
the data. This way, scattering profiles are coupled to each time point. A filter is then applied, to obtain
the scattering data within the filter range. Then, the data is integrated to find the integrated scattering
cross section, which is a measure of the scattering intensity. Lastly, the simulated camera captures this
scattering with the defined settings.
5.4 Conclusions on script development

The MNPBEM toolbox was used to create a look-up table to convert gap size over time data to scattering over
time. The influence of different filters was evaluated, after which was continued with 40 nm bandwidth filters.
Finally, a script was created that automatically gets the important variables from Laurens’s data and converts
it into scattering over time.
Lysanne Mol 27
SIMULATIONS
Chapter 6
Scattering Fluctuation of a
Particle-on-Film System over Time:
Simulations
In this chapter, the results from the simulations on scattering fluctuations over time are described. First, the
time-dependent gap size and scattering for 40 and 80 nm AuNPs is discussed, together with the possible tether
length resolution. Then, the effect of the standard deviation in nanoparticle size on this resolution is shown.
A solution on this problem of varying particle size is presented by introducing red-green ratio (RG-ratio) based
simulations. Lastly, the dynamic variation of RG-ratio over time is introduced as a way to distinguish the
different tether lengths.
6.1 Time-dependent scattering intensity for different tether lengths

The first simulations that were done were evaluations of the average scattering over time. The simulations were
done at 10 fps for 40 and 80 nm AuNPs with different tether lengths.
6.1.1 Simulating 40nm AuNPs

In Figure 6.1, the gap size over time and corresponding integrated scattering cross section over time for 40
nm AuNPs are displayed. It can be seen that for all tether lengths, the particles fluctuate around a certain
average gap size. The precise fluctuations can however only be seen at a higher frame rate. The average gap
size increases for increasing tether length, as is to be expected.
When looking at the integrated scattering cross section over time (which is a measure for the scattering intensity
over time), it can be seen that larger tether lengths result in a lower scattering intensity. This is due to the
decrease in plasmonic coupling that comes with increasing gap size. This decoupling results in a severe decrease
in overall scattering intensity.
6.1.2 Simulating 80nm AuNPs

The same simulation was done for 80 nm AuNPs, as can be seen in Figure 6.2. In Figure 6.2A, it can be seen
that the average gap size for each tether length is lower for 80 nm AuNPs than for 40 nm AuNPs. In Figure 6.2,
a major difference is seen compared to the 40 nm AuNPs, namely that here the scattering intensity increases
for an increasing tether length. This is due to the fact that larger particles decouple at gap sizes larger than 10
nm (as proven in Section 4.3), which is not reached in this case. The scattering intensity is here measured at the
green peak. When no decoupling takes place, the intensity in this area will increase for increasing gap size due
to the blue shift. Lastly, it is again seen that the scattering intensity is higher for larger particles. Previously
in this report, it was mentioned that larger particles diffuse slower and are therefore not necessarily better for
the purpose of this study. However, this difference in diffusion is only seen when the simulated camera is set at
a higher frame rate, for example 10,000 fps. It seems as if this is not needed, since the average scattering cross
section shows enough variation to distinguish the different tether lengths (as can be seen in Figure 6.2 and will
further be shown in the next section). Therefore, the difference in diffusion between 40 and 80 nm AuNPs is
not important, and 80 nm AuNPs can be used for their higher scattering intensity.
Lysanne Mol 28
SIMULATIONS
Figure 6.1: A: Gap size over time for 40 nm AuNPs at different tether lengths, as simulated by Laurens’s
model. It can be seen that larger tethers result in higher gap sizes. B: Integrated scattering cross section for 40
nm AuNPs at different tether lengths. The scattering cross section is integrated for wavelengths ranging from
580-620 nm. It can be seen that a larger tether length results in a lower scattering intensity.
Figure 6.2: A: Gap size over time for 80 nm AuNPs at different tether lengths, as simulated by Laurens’s
model. The same pattern can be seen as with the 40 nm AuNPs. B: Integrated scattering cross section for 40
nm AuNPs at different tether lengths. The scattering cross section is integrated for wavelengths ranging from
580-620 nm. It can be seen that an increase in tether length results in an increase in scattering intensity.
6.1.3 Relation between tether length and mean scattering intensity

To make the relation between the scattering intensity and the scattering over time more clear, the mean
scattering cross section is plotted against different tether lengths in Figure 6.3. The data is fitted using the
curve fitting toolbox in MATLAB with R2 = 0.9882. It can be seen that there is a close to linear relationship
between the tether length and the scattering intensity, where even tether length differences of 3-4 bp are visible.
Since these simulations are based on randomized movement of the gold nanoparticles, it is important to know
whether the mean scattering intensity is relatively constant. After all, when the resolution on which two tethers
can be distinguished from each other is determined by the mean scattering intensity, very little variation is
allowed. To evaluate this, a simulation with 80 nm beads on a 50 bp tether was performed three times, after
which the mean scattering cross section was determined. The simulated mean scattering turned out to be
1.1286 ± 0.0005 ∗ 106 nm3 . This deviation is far less than the difference in scattering for two tether lengths with
a 3-4 bp difference, thus the randomized movement should have no influence on the resolution.
Lysanne Mol 29
SIMULATIONS
Figure 6.3: The mean scattering cross section for 80 nm AuNPs at tether lengths ranging from 50 to 85 nm.
The data is fitted with the line f (x) = 1652x + 9.447 ∗ 105 , where R2 = 0.9882. It can be seen that differences
in tether length of 3-4 bp are visible.
6.2 Influence of standard deviation of particle size

The results described thus far have been very promising for the possibility of multiplex testing: the minimal
tether length difference needed to distinguish two signals from each other based on scattering intensity only
needed to be a few bp. However, one factor has yet to be considered, which is the standard deviation in
nanoparticle size. According to the manufacturer, the size of 80 nm AuNPs ranges from 77.0-85.0 nm [33]. Since
the scattering intensity strongly depends on the particle size, especially for larger particles, it was worthwhile to
evaluate the influence of this standard deviation on the resolution. For this, simulations were done using 80 and
82 nm AuNPs, to get an insight what the influence might be. In Figure 6.4A, it is seen that the nanoparticle
size has very little influence on the mean gap size that is reached for the different tether lengths. This was to
be expected, since the main influence on the average position of the nanoparticle (and thereby the mean gap
size) is mainly determined by the tether and less by the nanoparticle itself (as is explained in more depth in
Laurens’s thesis [10]). However, when looking at Figure 6.4B, one can see that the difference in mean scattering
cross section is very severe. Since the inaccuracy means that many particle sizes between 80 and 82 nm will
occur, as well as several sizes outside of this range, it is impossible to find the right combination of tether
length and particle size merely based on scattering intensity. The cause for this can be seen in Figure 6.4C,
where the scattering plots are given for 77-85 nm AuNPs. Due to the strong relation between the AuNP size
and the scattering intensity (see Section 4.2), a small increase in particle size results in a substantial increase
in scattering cross section.
Lysanne Mol 30
SIMULATIONS
Figure 6.4: A: The mean gap size for 80 and 82 nm AuNPs with different tether lengths. B: The mean scattering
cross section for 80 and 82 nm AuNPs with different tether lengths. C: The scattering plots for 77-85 nm AuNPs
located 6 nm from the surface. The lowest graph corresponds to 77 nm AuNPs, the top graph to 85 nm AuNPs.
It can be seen that the small change in nanoparticle size has great influence on the scattering plot, which results
in a large increase in mean scattering cross section.
6.3 Red-green ratio for different tether lengths

When looking at Figure 6.4C, one can see that the deviation in AuNP size results in the scattering plot shift-
ing. This led the hypothesis that the effect of the nanoparticle size might be less severe on the red-green ratio
(RG-ratio) than on the mean scattering cross section, since the increase in scattering cross section seemed to
be the same for both the red and the green peak. Thus, the script from Appendix H was altered to be able to
measure using two filters. The first simulations used a filter for the red peak ranging from 748-780 nm and for
the green peak from 580-620 nm. The results of this are seen in Figure 6.5. It can be seen that the difference
in RG-ratio for both AuNP sizes is less than before. It also seems as if the difference becomes less when the
tether length increases. This is uncertain however, since the simulation with 80 nm AuNPs was done with far
more tether lengths than the simulation with 82 nm AuNPs.
The next hypothesis was that a larger filter bandwidth would decrease this difference within the particle
standard deviation. After all, the larger the surface under the graph that is measured, the smaller the influence
of a certain variation. Therefore, the simulation was repeated using filters ranging from 700-820 nm and 540-660
nm. The results are seen in Figure 6.6. It can be seen that the hypothesis was correct and that the difference
in RG-ratio is indeed smaller for this larger filter bandwidth.
The parameter that is observed to distinguish the different signals from each other is changed from the scattering
intensity to the RG-ratio. As a result, the resolution has worsened. However, even with the variation in
nanoparticle size, a difference in 10 bp for a tether should be distinguishable. To validate this, bars were added
to this plot based on the standard deviation of the RG-ratio. This standard deviation is a result of the dynamic
variation in particle position. Figure 6.7A indeed shows that a difference in 10 bp is distinguishable for tether
lengths between 50 and 70 bp.
The same figure was made for a camera that measures at 1000 fps, as can be seen in Figure 6.7B. A large
increase in standard deviation can be seen, which results in the fact that the signal of all tether lengths overlap.
Lysanne Mol 31
SIMULATIONS
Figure 6.5: Red-green ratio for 80 and 82 nm AuNPs with different tether lengths. The scattering intensity is
determined for wavelengths reaching from 580-620 nm and 740-780 nm. It can be seen that the RG-ratio for
82 nm particles is generally higher.
Figure 6.6: Red-green ratio for 80 and 82 nm AuNPs with different tether lengths. The scattering intensity is
determined for wavelengths reaching from 540-660 nm and 700-820 nm.
This occurrence was also mentioned by Visser et al. [20]. The cause of this increase in standard deviation is
the fact that with a higher fps, more movement is measured. Therefore, more dynamic variation is measured,
since less movement is averaged out. Thus, measuring with a low fps is preferred when one wants to evaluate
the mean RG-ratio. However, it is uncertain if this mean and standard deviation is constant for multiple
measurements of the same AuNP-tether combination. For this, the standard deviation of the mean RG-ratio
of multiple simulations of the same AuNP-tether should be evaluated. Due to time constraints, this was not
possible.
6.4 Dynamic variance of RG-ratio

Thus far, it was shown that theoretically two signals can be distinguished from each other by the mean scat-
tering cross section if the difference is only 3-4 bp. However, when particle heterogeneity is considered, a 10
bp difference in tether can only be measured when a very wide filter and low fps is used. Even in this case, it
was uncertain if multiple measurements will lead to the same mean RG-ratio. In other words, it was unknown
if this proposed method leads to significant results. Therefore, a new approach was needed.
From Laurens’s model, it was noted that an increase in tether size not only resulted in an increase in average
Lysanne Mol 32
SIMULATIONS
Figure 6.7: Mean RG-ratio for 80 nm AuNPs at different tether lengths measured at 10 fps (figure A) and 1000
fps (B). The ”error” bars indicate the dynamic variation in RG-ratio due to particle movement. It can be seen
that a higher fps results in a larger standard deviation, since less movement is averaged out.
gap size, but also in an increase in standard deviation (or variance) of this gap size [10]. It was also noted that
the standard deviation of this variance was small. Therefore, the hypothesis was that this variance might be a
more characteristic measure for the different tether lengths.
To prove this hypothesis, several simulations were performed for 80 nm AuNPs with different tether lengths.
Each measurement was repeated 3 times to get an idea of the variance within these measurements. The output
of each measurement is the RG-ratio over time. This ratio fluctuates due to the movement of the particle.
Since this movement depends on the tether length, the fluctuations depend on this tether length. This dynamic
fluctuation is calculated by taking the standard deviation of the RG-ratio over time for the entire measurement.
This is done for all three measurements. Since the movement is random, a slight variation in this dynamic
fluctuation is to be expected. Therefore, the standard deviation in dynamic variance is determined from these
three measurements. Lastly, a two-sampled t-test is performed to prove that the difference in dynamic fluctua-
tion (including the standard deviation) is significant. For this, the null hypothesis was that the mean dynamic
variance values of the different tether lengths are the same and any difference is caused by chance. The alter-
native hypothesis was that the mean values are not equal and that the difference is significant.
The figures mentioned below show the dynamic fluctuation of the RG-ratio for the different tether lengths,
together with the standard deviations of these fluctuations. Then, using the two-sampled t-test, the value for
p is determined to indicate the significance. This value of p is given with a certain number of asterisks. The
number of asterisks relates to p as follows: * p = 0.05, ** p = 0.01, *** p = 0.001, >*** p = 0.0001.
Figure 6.8: The dynamic fluctuation of the RG-ratio for different tether lengths, measured at A) 10 fps, B)
1000 fps, C) 10,000 fps. The significance of the difference in fluctuations is determined using the two-sampled
t-test. The difference is not significant when measured at 10 fps. The highest significance is obtained when
measuring at 10,000 fps.
The first simulations were done using the same wide filter with a bandwidth of 120 nm. In Figure 6.8A the
measurements are seen for a 10 fps camera, and in Figure 6.8B for 1000 fps. It can be seen that no significant
difference between the different tethers is seen when measuring at 10 fps. However, when measuring at 1000
Lysanne Mol 33
SIMULATIONS
fps, the two-sampled t-test shows that all tethers can be distinguished from each other. This can be explained
by the fact that the fps determines how much the movement was averaged. As shown before in Figure 6.1
and Figure 6.2, very little variation is seen when measuring at 10 fps. This also means that any differences in
movement between the varying tether lengths are not seen, since these movements are averaged out. However,
when measuring at 1000 fps, less averaging takes place, such that the difference in movement becomes more
visible. this effect is seen in Figure 6.8 as a higher dynamic fluctuation of the RG-ratio and a smaller standard
deviation of this fluctuation. The final simulation was done at 10,000 fps, as shown in Figure 6.8C. Here, the
smallest value for p was obtained, indicating that the most significant difference is found at this frame rate.
However, the highest frame rate is not necessarily the best option, since this also results in more Brownian
noise [10, 20].
To take into account the particle heterogeneity, this test was repeated using 80 and 82 nm particles. Figure 6.9A
shows the results using the same filter as before and a camera measuring at 1000 fps. It can be seen that the
standard deviation has increased, but that the difference between the different tether lengths is still significant.
The same test was repeated for a smaller filter with a 40 nm bandwidth, seen in Figure 6.9B. Just as in previous
simulations, the smaller filter resulted in a more substantial difference in RG-ratio due to a relatively larger
difference in scattering. The difference between the tether is still significant, but with a higher value for than
for the wider filter. Therefore, a larger bandwidth is recommended.
Figure 6.9: The dynamic fluctuations of the RG-ratio for different tether lengths, measured at 1000 fps using A)
a 120 nm bandwidth filter, B) a 40 nm bandwidth filter. A smaller filter results in a more significant difference
in dynamic fluctuation for different tether lengths.
6.5 Conclusions on scattering fluctuation over time

In this chapter, the simulations that were done on scattering fluctuation over time were described. First, the
difference in gap size over time and scattering over time for 40 and 80 nm AuNPs was studied. After this, it
was concluded that the best results were obtained using 80 nm AuNPs, since these particles have the highest
scattering intensity. The fact that 80 nm AuNPs move slower did not create an issue, since measuring the
mean scattering cross section at 10 fps showed enough difference between the different tethers. However, then
it was shown that the small variation in nanoparticle size did lead to issues, since this variation resulted in a
substantial variation in scattering intensity. This was solved by measuring the RG-ratio instead of the mean
scattering cross section. The problem was further reduced by introducing filters with wider bandwidths. It was
however not proven that the observed difference in mean RG-ratio was significant. It was demonstrated that
this did result in significant differences for the different tether lengths, which was proven using the two-sampled
t-test. It was still recommended to use a wide filter, but in this case a higher frame rate also lead to better
results.
Lysanne Mol 34
CHAPTER 7. DISCUSSION AND RECOMMENDATIONS
Chapter 7
Discussion and Recommendations
In this chapter, several points that possibily influence the simulated results are brought to the attention. The
possible effects of these factors are mentioned, together with suggestions on how to improve the simulations.
Besides that, suggestions are given for further experiments to validate the simulated results.
7.1 Discussion on simulated results

All simulations are done to represent the experimental situation as close as possible. Still, some points of
improvement were found.
The first aspect that will possibly result in a difference in the experimental results compared to the simulated
results is the fact that the simulations assume that all scattered light is actually captured by the microscope.
However, the dark-field microscope that is used in the experiments excites the particles at a large angle, but
captures the light directly above the particle. This is not optimal, since the light is scattered at an large angle,
especially for the longitudinal dipole. Therefore, it is recommended to repeat the simulations with this in mind,
since this will likely affect the measured scattering intensity. This might even influence the RG-ratio, since the
red peak is the result of the longitudinal dipole peak, which is the dipole that will scatter light at a large angle.
The green peak however scatters more light directly on the detector. Therefore, the RG-ratio could be lower
in practice than in theory, since not all the red light will be captured.
Another point on which the simulations might differ from the experimental results is when measuring with
gap sizes smaller than 1 nm. At these gap sizes, quantum tunneling and electron nonlocality will occur [13, 34],
which are not considered in the MNPBEM toolbox. For the first part of the thesis it was therefore avoided
to simulate at gap sizes this small. However, for the second part this was not possible, since the results from
Laurens’s model showed that gap sizes this small will occur. Unfortunately, there is no possibility in the
MNPBEM toolbox to implement the quantum tunneling and electron nonlocality effects. Thus, these effects
must be simulated outside of the toolbox in MATLAB.
For the simulations described in the second part of the thesis, more assumptions are done that might in-
fluence the accuracy of the results. One of these is the resolution of the look-up table. Since the sensitivity
of the camera was uncertain, an educated guess was taken to determine the resolution of the table. The steps
taken in the table might be larger than the steps that are measurable by the camera, which will lead in an
difference in simulated and measured dynamic variance.
The last assumption had to do with the simulated filters. Since the available filters were unknown and the
bandwidth of many dark-field microscope filters are not given, fictional filters were used in the simulations. This
way, the effect of the filters could be studied, without knowing the exact filters that were available. However,
this also means that there is the possibility that the optimal results are obtained using a filter that is not
commercially available. It is of course recommended to find a filter as close to the simulated optimal filter,
however, this is not always possible. A different filter will result in a difference in simulated and experimental
results and will also influence the resolution on which the difference in tether length can be determined, as
shown in Section 6.4. The filters were also assumed to have perfect rectangular filter windows. Since this is
not realistic, this might influence the results. When commercially filters are chosen, the filter windows of these
filters can be recreated in MATLAB to have a better representation of the filters.
Lysanne Mol 35
7.2 Suggestions for further experiments

The first part of this thesis consisted of experiments that have been done multiple times before, either by
simulations or by experiments. The relations between the influencing factors and the optical response were
in accordance with literature. However, to validate correctness of the values, experiments with this specific
system are needed. For the second part, experimental validation of the current simulated results is also needed.
To give proper recommendations on these experiments, first the possible imaging techniques that are used in
literature for this purpose are reviewed.
7.2.1 Imaging techniques

Several different techniques can be used for imaging plasmon resonance based on literature. The first option
is using a dark field microscope with a CCD camera and filters, or with a color camera. The CCD enables
measuring at higher frame rate. The issue with this technique is that only one wavelength can be measured,
or the total scattering intensity (in case a CCD is used without any filter). When two wavelengths need to be
measured, a beam splitter is needed. A second option uses a hyperspectral microscope, which is the technique
used by Armstrong et al. [22]. This enables measuring the scattering intensity throughout the full visible
spectrum. A third option uses a camera with a spectrometer combined with a CCD or color camera, as used
by Mock et al. [16]. Just as with the hyperspectral microscope, the spectrometer enables measuring the full
spectrum. By using a beam splitter, the overall image can be created using a CCD or color camera. Which of
these options is recommended depends on the experiment, as will be explained later.
7.2.2 Experiments to validate the effect of influencing factors

The effect of the incident angle cannot be validated, since the incident angle of the light from the microscope
is set by its NA. However, the experimental results can be compared with a simulation done for an incident
angle of 60 degrees, since this matches the incident angle of the objective. This can be done by using 40 nm
of 80 nm AuNPs bound to a DNA hairpin where the distance between the AuNP and the surface (in other
words, the height of the hairpin when it is folded) is known. It is recommended to validate when the hairpin
is folded, since this reduces the movement of the particle, so that the scattering signal is relatively constant.
When it is not possible to obtain a certain gap size this way, it can be considered to validate the results using
a spacer layer. Spacer layers are more often used to create a constant distance between the particle and the
film, for example by Armstrong et al. [22] and Mock et al. [16]. Please note however that in this case, the
spacer layer should be added to the defined layers in MATLAB. The experiments can then be compared with
simulations that have the same input for incident angle, gap size and AuNP size. For the best validation, it is
recommended to evaluate the full spectrum, which means using a hyperspectral microscope or a spectrometer.
If this is not possible, a CCD camera can be used to compare the overall intensity and a color camera can
be used to compare the intensities at the green and red wavelength area. This way, one can still compare the
overall intensity and the relative intensities of the different peaks.
The effect of AuNP size can easily be validated by measuring the scattering intensity for 20, 40, 60 and
80 nm AuNPs using a CCD camera. The intensities for the varying AuNP sizes can then be compared the
the theoretical relation. Note however that the gap size should be known and constant in order to compare
the results to the simulations. Therefore, it is again recommended to perform the measurements with folded
hairpins or a spacer layer, since this results in a more constant gap size. When one wants to evaluate the full
spectrum, a hyperspectral microscope or spectrometer is again recommended.
Lastly, the effect of gap size can be validated by measuring the scattering spectrum (using a hyperspectral mi-
croscope or spectrometer) or the scattering intensity (using a CCD camera) for different DNA hairpin lengths.
When the equipment to evaluate the full spectrum is not available, a color camera can used to get an idea of
the relative intensities of the different scattering peaks. By measuring both folded and unfolded hairpins, a
wider range of gap sizes can be measured. The issue with unfolded hairpins however is that the gap size will
not be constant, due to movement of the AuNP. In this case, the average gap size can be determined using
Laurens’s model, to get a rough estimation of the gap size. Another solution is again to use a spacer layer of
varying thickness, to obtain different gap sizes.
Lysanne Mol 36
7.2.3 Experiments to validate the scattering fluctuation over time

Since very little comparable research can be found to validate the simulations for scattering fluctuation over
time, it is important to experimentally validate the results. For this, it is most important to validate the
time-traces, since these are the basis of the other simulations, and the dynamic variance of the RG-ratio. It is
recommended to perform the experiments using a high-speed camera with red and green filters. For the filters,
a large bandwidth is recommended (e.g. 120 nm). The measurements should be performed for 2 seconds, such
that the total measurement time is equal to many times the relaxation time. For the dynamic variance of the
RG-ratio, it should not matter if the red and green wavelengths are measured at the same time or not due to the
long measuring time. For the time-traces however, it is beneficial to be able to measure both simultaneously,
to be able to compare the effect of the movement for both resonance peaks. This can be done using a beam
splitter.
When a certain experimental set-up is chosen, it is recommended to determine the minimal variation in scatter-
ing intensity that can be measured. This minimal change can than be used to improve the look-up table, since
the current resolution of this table is determined with an educated guess. By improving this look-up table, the
simulated results will be more accurate.
7.3 Suggestions on further simulations

Many simulations have been done during this research. However, further simulations can be used to obtain
results that are better comparable to experimental results. Other simulations are needed to further research
some of the observations that were done in this thesis, especially in the second part of this research. Therefore,
some suggestions are done on simulations that can be done for this end.
7.3.1 Further simulations on influencing factors

As said before, the simulations assumed that all the scattered light was captured by the microscope, which is
not the case. This will results in the fact that simulated intensity and RG-ratio will differ from the experimental
results. Therefore, it is recommended to improve the simulations by taking this into account. The exact effect
on the different results can be simulated by only evaluating the scattering intensity for the scattering that
occurs within the range of the angles that are measured by the microscope. This might however not be possible
using the MNPBEM toolbox, since this toolbox does not consider the scattering pattern, it just calculates all
the scattering that occurs. The solution for this is to research the scattering pattern for a particle, evaluate
what percentage of the scattered light could be caught by the camera and implementing this percentage in the
MNPBEM calculations. When this is done, the simulations can be redone and compared to the experiments for
validation. Something similar has been done by Visser et al. [20], where the efficiency of the imaging system
was also estimated.
7.3.2 Further simulations on scattering fluctuation over time

During the simulations, one major issue turned out to be the standard deviation in particle size. However, due
to time constrains, this factor was only studied briefly. It is recommended to continue the simulations with
multiple AuNP sizes within the range of 77-85 nm to further study the effect of this on the resolution. With
the current simulations, it is unclear if the difference in optical response between the 80 and 82 nm AuNPs
becomes less with increasing tether length, or if this is just the result of a lack of data. Therefore, in future
simulations it is important to include many tether lengths, just as was done for 80 nm AuNPs in this research,
such that the linear relationship can be made clear. It is also needed to evaluate the effect of including more
particle sizes on the significance of the difference dynamic variance: it was seen that adding 82 nm particles
in the simulations resulted in an increase in standard deviation of the dynamic variance. It might be the case
that adding all the different particle sizes within the 77-85 nm range results in such an increase in standard
deviation that the difference in dynamic variance is no longer significant.
The resolution can also be further evaluated by testing more tether lengths. Due to time constraints, the
simulations for dynamic variation in RG-ratio have only been done for 50, 60 and 70 bp tethers. It is recom-
mended to test with even longer tethers to see if the resolution of 10 bp still leads to significant results. Smaller
differences in tether length can also be tested, to see if the resolution can be improved even more.
One last suggestion for improving the resolution is by changing the filter window. The filters were now chosen
such that the middle of the window was around the peaks in the scattering plot. However, at wavelengths just
beside those peaks, the difference in scattering due to particle heterogenicity is less, as seen in Figure 6.4C.
This may result in a higher resolution.
Lysanne Mol 37
Besides different particle sizes within the standard deviation range, even more particle sizes can be evalu-
ated aside from 40 and 80 nm. After all, the effect of the variation in particle size will be less significant for
smaller particles, since the scattering intensity is related to particle size to the power 4.8. This fact argues for
using smaller particles to create a better resolution. Another advantage is that 40 nm particles have a lower
relaxation time, which results in lower measurement times. However, the trade-off is that the intensity will
be smaller, so with experiments it should be evaluated what the minimum intensity is needed to get proper
results. Besides that, 40 nm AuNPs will most likely decouple due to the average gap size being higher than
5 nm for all tether lengths. This could influence the dynamic variance, which is undesirable. Simulations for
varying particle sizes below 80 nm can predict the impact of the particle heterogeneity, scattering intensity and
(possible) decoupling to obtain the optimal particle size. Particles larger than 80 nm are only recommended
when the intensity forms an issue, since the variation in particle size will lead to even larger effects in the
scattering results, thereby worsening the resolution.
Lysanne Mol 38
CHAPTER 8. CONCLUSION
Chapter 8
Conclusion
In this thesis, a particle-on-film system is simulated to evaluate the effect of several influencing factors as well as
simulate the scattering fluctuation over time. This assignment was split into two parts. The research question
that was stated for the first part was: ”What is the effect of gap size, AuNP size and incident angle on the
scattering cross sections and near-field plots of AuNPs on a gold film?”
The effect of the incident angle was simulated first. It was found that for a larger incident angle, the longitudinal
dipole peak increases. This can be explained by the fact that the different light polarizations excite the different
dipole states. The incident angle determines the ratio between the polarizations and thereby the intensity of
the different dipole peaks in the scattering. In the near-field, it was found that an increase in incident angle
lead to an increase in maximum local field enhancement, with a plateau above 50 degrees. Further simulations
were done with an incident angle of 60 degrees since this matched both the optimal angle according to the
simulations and the incident angle of the dark-field microscope used in the simulations.
It was also shown that an increase in AuNP size resulted in an increase in local field enhancement in the
near-field. The relation between the total scattering in the far-field and the AuNP size was determined to be
σsca ∼ R4.8 for a gap size of 2 nm. This relation approached σsca ∼ R6 for an increasing gap size. Further
simulations were continued with particle sizes of 40 and 80 nm, taking in mind both the scattering intensity
and the diffusion coefficient.
Lastly, the blue-shift as a result of an increase in gap size was proven for 40 and 80 nm AuNPs. It was concluded
that this blue-shift took place at smaller gap sizes for smaller particles. This is because the coupling between
the particle and the film is stronger for larger particles.
The goal of the second part of the project was to evaluate the scattering fluctuation over time as a result
of the particle movement over time. The ultimate goal was to find a way to distinguish two different signals
from each other based on tether length. It was finally concluded that this was best done using 80 nm AuNPs
due to their high scattering intensity. The relaxation time of the system was found to be in the milliseconds
range, after which was decided to measure for 2 seconds. Several different methods for distinguishing the
two particles were tried, and finally the best system turned out to be the difference in dynamic fluctuation of
the RG-ratio. This was best measured using a high frame rate (>1000 fps) and a wide filter (120 nm bandwidth).
Lastly, some recommendations were given for future simulations and experiments. It was recommended to
validate the simulated results using a system to evaluate the full spectrum (hyperspectral microscope or spec-
trometer) and/or using a system to evaluate the scattering intensity (CCD or color camera). For future
simulations, it is recommended to take into account that not all the scattered light reaches the detector, to
improve the gap size look-up table and to simulate more AuNP size including their standard deviation.
When the particle-on-film system in this thesis is optimized and tested further, it is likely that differences
in 10 bp tether lengths will be distinguishable, which would enable multiplexed sensing. Hereby, the goal of
hmDNA detection on a chip to test for cancer types in an early stage is one step closer.
Lysanne Mol 39
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Lysanne Mol 42
Appendices
A. Script for far-field validation using simulated data

1 %% S c r i p t f o r v a l i d a t i n g f a r − f i e l d p l o t s u s i n g s i m u l a t e d data
2 % Date : 02−06−2020
3 % S c r i p t from Armstrong a l t e r e d by Lysanne Mol
4
5 %% S e t p a r a m e t e r s
6 material = 'gold ' ; % m a t e r i a l o f t h e NP and f i l m
7 diameter = 100; % d i a m e t e r o f t h e NP [nm]
8 film thickness = 45; % t h i c k n e s s o f ' m a t e r i a l ' f i l m [nm]
9 spacing = 5; % t h i c k n e s s o f s p a c e r l a y e r [nm]
10 t h e t a = 70 * p i / 1 8 0 ; % i n c i d e n t a n g l e [ rad ]
11 lambda = l i n s p a c e ( 5 0 0 , 9 0 0 , 1 0 0 ) ; % w a v e l e n g t h s o f l i g h t [nm]
12
13 %% D i e l e c t r i c f u n c t i o n s
14 % n med = r e f r a c t i v e i n d e x environment
15 % n subs = r e f r a c t i v e index s u b s t r a t e
16 % n gap = r e f r a c t i v e i n d e x s p a c e r l a y e r
17
18 n med =1; n s u b s = 1 . 5 2 ; n gap = 1 . 5 ;
19 e p s t a b = { e p s c o n s t ( n med ˆ2 ) , e p s t a b l e ( [ m a t e r i a l ' . dat ' ] ) , e p s c o n s t ( n s u b s ˆ2
) , e p s t a b l e ( [ m a t e r i a l ' . dat ' ] ) , e p s c o n s t ( n gap ˆ2 ) , e p s t a b l e ( [ ' chromium .
dat ' ] ) } ;
20
21 %% o p t i o n s f o r BEM s i m u l a t i o n
22
23 % d e f a u l t o p t i o n s f o r l a y e r s t r u c t u r e
24 op = l a y e r s t r u c t u r e . o p t i o n s ;
25
26 % l o c a t i o n o f i n t e r f a c e o f s u b s t r a t e
27 z t a b = [−1 * ( d i a m e t e r / 2 ) , −1 * ( d i a m e t e r /2 + s p a c i n g ) , −1 * ( d i a m e t e r /2 +
s p a c i n g )− f i l m t h i c k n e s s , −1 * ( d i a m e t e r /2 + s p a c i n g )− f i l m t h i c k n e s s − 5 ] ;
28
29 l a y e r = l a y e r s t r u c t u r e ( epstab , [ 1 , 5 , 4 , 6 , 3 ] , ztab , op ) ;
30 op = bemoptions ( ' sim ' , ' r e t ' , ' i n t e r p ' , ' curv ' , ' l a y e r ' , l a y e r ) ;
31
32 % P p o l e x c i t a t i o n from above
33 d i r = [ s i n ( t h e t a ) , 0 , − c o s ( t h e t a ) ] ;
34 p o l = [ 1 * c o s ( t h e t a ) , 0 , 1 * s i n ( t h e t a ) ] ;
35 exc1 = planewave ( pol , d i r , op ) ;
36
37 % S p o l e x c i t a t i o n from above
38 d i r 2 = [ s i n ( t h e t a ) , 0 * t h e t a , − c o s ( t h e t a ) ] ;
39 p o l 2 = [ 0 * t h e t a , 1 * t h e t a , + 0 * t h e t a ] ;
40 exc2 = planewave ( pol2 , d i r 2 , op ) ;
41
42
43 %% PARTICLE DEFINITION
44
Lysanne Mol 43
45 % i n i t i a l i z e n a n o s p h e r e
46 p = t r i s p h e r e ( 2 0 0 , d i a m e t e r ) ;
47
48 % This command w i l l c r e a t e t h e system t o be c a l c u l a t e d − i n p i s now t h e
p a r t i c l e s ( and i t s d i m e n s i o n s ) + a l l m a t e r i a l s and t h e i r p o s i t i o n i n t h e
system
49 p = c o m p a r t i c l e ( epstab , { p } , [ 2 , 1 ] , 1 , op ) ;
50
51 % t a b u l a t e d Green f u n c t i o n s
52 % For t h e r e t a r d e d s i m u l a t i o n we f i r s t have t o s e t up a t a b l e f o r t h e
c a l c u l a t i o n o f t h e r e f l e c t e d Green f u n c t i o n . % % % This p a r t i s u s u a l l y s l o w
and we t h u s compute GREENTAB o n l y i f i t has not been computed b e f o r e .
53
54 % automatic g r i d f o r t a b u l a t i o n
55 tab = t a b s p a c e ( l a y e r , p , ' nz ' , 50 ) ;
56 % Green f u n c t i o n t a b l e
57 g r e e n t a b = c o m p g r e e n t a b l a y e r ( l a y e r , tab ) ;
58 % precompute Green f u n c t i o n t a b l e
59 g r e e n t a b = s e t ( g r e e n t a b , lambda , op ) ;
60 op . g r e e n t a b = g r e e n t a b ;
61
62 %% BEM s o l v e r
63
64 % s e t up BEM s o l v e r
65 bem = b e m s o l v e r ( p , op ) ;
66
67 % t h e l o o p c a l c u l a t e s s u r f a c e c h a r g e s and s c a t t e r i n g c r o s s s e c t i o n i n nm2
68 f o r i e n = 1 : l e n g t h ( lambda )
69 % s u r f a c e charge
70 s i g 1 = bem \ exc1 ( p , lambda ( i e n ) ) ;
72 % scattering cross section
73 s c a 1 ( i e n , : ) = abs ( exc1 . s c a ( s i g 1 ) ) ;
75 end
76
77 %% T o t a l s c a t t e r i n g c r o s s s e c t i o n
78 s c a t o t = s c a 1 + s c a 2 ;
Lysanne Mol 44
B. Script for near-field validation using simulated data
2 % Date : 02−06−2020
3
8 spacing = 5; % t h i c k n e s s o f s p a c e r l a y e r [nm]
10 lambda = 8 2 0 ; % w a v e l e n g t h s o f l i g h t [nm]
11
16
17 n med =1; n s u b s = 1 . 5 2 ; n gap = 1 . 5 ;
dat ' ] ) } ;
19
s p a c i n g )− f i l m t h i c k n e s s , −1 * ( d i a m e t e r /2 + s p a c i n g )− f i l m t h i c k n e s s − 5 ] ;
22
25
26 % S e t up l a y e r s t r u c t u r e
29
30 %% P a r t i c l e d e f i n i t i o n
31 p = t r i s p h e r e ( 2 0 0 , d i a m e t e r ) ;
33
34 %% I n i t i a l i z i n g planewave
35
37 d i r = [ s i n ( t h e t a ) , 0 , − c o s ( t h e t a ) ] ;
38 p o l = [ 1 * c o s ( t h e t a ) , 0 , 1 * s i n ( t h e t a ) ] ;
40
42 d i r 2 = [ s i n ( t h e t a ) , 0 * t h e t a , − c o s ( t h e t a ) ] ;
43 p o l 2 = [ 0 * t h e t a , 1 * t h e t a , + 0 * t h e t a ] ;
45
46 %% Green f u n c t i o n s
47 % Green f u n c t i o n s
48 % c r e a t e mesh on which f i e l d i s e v a l u a t e d
49 [ x , z ] = meshgrid ( l i n s p a c e ( − 6 0 , 6 0 , 161 ) , l i n s p a c e ( −60, 6 0 , 201 ) ) ;
50 pt = compoint ( p , [ x ( : ) , 0 * x ( : ) , z ( : ) ] , op ) ; % p l a c e o b j e c t i n mesh
51 tab = t a b s p a c e ( l a y e r , p , pt , ' nz ' , 5 ) ;
53 g r e e n t a b = s e t ( g r e e n t a b , lambda , op , ' w a i t b a r ' , 1 ) ;
55
Lysanne Mol 45
56 %% s o l v e BEM e q u a t i o n s
58
59 % c a l c u l a t e s u r f a c e charge f o r given wavelengths
60 s i g 1 = bem \ exc1 ( p , lambda ) ;
61 s i g 2 = bem \ exc2 ( p , lambda ) ;
62
63 %% e m i s s i o n p a t t e r n
64 % angles
65 p h i = r e s h a p e ( l i n s p a c e ( 0 , 2 * pi , 301 ) , [ ] , 1 ) ;
66
67 % directions for emission
68 emdir = [ c o s ( p h i ) , 0 * phi , s i n ( p h i ) ] ;
69
70 % s e t up spectrum o b j e c t
71 s p e c = spectrum ( emdir , op ) ;
72
73 %% computation o f e l e c t r i c f i e l d
74 emesh = m e s h f i e l d ( p , x , 0 , z , op , ' m i n d i s t ' , 0 . 5 , 'nmax ' , 2000 ) ;
75
76 % i n d u c e d and incoming e l e c t r i c f i e l d
77 e1 = emesh ( s i g 1 ) + emesh ( exc1 . f i e l d ( emesh . pt , lambda ) ) ;
78 e2 = emesh ( s i g 2 ) + emesh ( exc2 . f i e l d ( emesh . pt , lambda ) ) ;
79
80 % norm o f e l e c t r i c f i e l d
81 e e 1 = s q r t ( dot ( e1 , e1 , 3 ) ) ;
82 e e 2 = s q r t ( dot ( e2 , e2 , 3 ) ) ;
83
84 % add f i e l d s o f both p o l a r i z a t i o n s
85 ee = ee1 + ee2 ;
Lysanne Mol 46
C. Script for far-field validation using experimental data
2 % Date : 02−06−2020
3 % S c r i p t from Armstrong a l t e r e d by Lysanne Mol
4 % The s c r i p t i s a l t e r e d t o be a b l e t o t e s t m u l t i p l e gap s i z e s , which was
5 % n e c c e s a r y t o r e c r e a t e t h e data from Mock e t a l .
6
11 spacingrange = [ 3 . 8 7.2 14.3 2 2 . 3 ] ; % t h i c k n e s s o f s p a c e r l a y e r [nm]
13 e n e i = l i n s p a c e ( 500 , 900 , 40) ; % w a v e l e n g t h s o f l i g h t [nm]
14
19
20 n med =1; n s u b s = 1 . 5 2 ; n gap = 1 . 5 4 ;
dat ' ] ) } ;
22
23 %% o p t i o n s f o r BEM s i m u l a t i o n
24
25 % default options for layer structure
27
28 % c r e a t e a r r a y s t o s a v e s c a t t e r i n g data . The f i r s t and s e c o n d d i m e n s i o n
29 % s a v e data f o r each wavelength , t h e t h i r d d i m e n s i o n s a v e s data f o r each
30 % gap s i z e .
31 s c a 1 = NaN( l e n g t h ( e n e i ) , s i z e ( d i r , 1 ) , l e n g t h ( s p a c i n g r a n g e ) ) ;
32 s c a 2 = NaN( l e n g t h ( e n e i ) , s i z e ( d i r 2 , 1 ) , l e n g t h ( s p a c i n g r a n g e ) ) ;
33 for i s = 1: length ( spacingrange )
34 spacing = spacingrange ( i s )
35 % location of interface of substrate
s p a c i n g )− f i l m t h i c k n e s s , −1 * ( d i a m e t e r /2 + s p a c i n g )− f i l m t h i c k n e s s
− 5 ] ; % z t a b = −1 * ( d i a m e t e r /2 + s u b s d i s t a n c e ) ;
37
40
42 dir = [ sin ( theta ) , 0 , − cos ( theta ) ] ;
43 pol = [ 1 * cos ( theta ) , 0 , 1 * sin ( theta ) ] ;
45
47 d i r 2 = [ s i n ( theta ) , 0 * theta , − cos ( theta ) ] ;
48 p o l 2 = [ 0 * t h e t a , 1 * t h e t a , + 0* t h e t a ] ;
50
51
53
54 % i n i t i a l i z e nanosphere
Lysanne Mol 47
55 p = t r i s p h e r e ( 200 , diameter ) ;
56
57 % This command w i l l c r e a t e t h e system t o be c a l c u l a t e d − i n p i s now t h e
p a r t i c l e s ( and i t s d i m e n s i o n s ) + a l l m a t e r i a l s and t h e i r p o s i t i o n i n t h e
system
59
60 % t a b u l a t e d Green f u n c t i o n s
61 % For t h e r e t a r d e d s i m u l a t i o n we f i r s t have t o s e t up a t a b l e f o r t h e
c a l c u l a t i o n o f t h e r e f l e c t e d Green f u n c t i o n . % % % This p a r t i s u s u a l l y
s l o w and we t h u s compute GREENTAB o n l y i f i t has not been computed
before .
62
68 g r e e n t a b = s e t ( g r e e n t a b , e n e i , op ) ;
70
72
75
76 % t h e l o o p c a l c u l a t e s s u r f a c e c h a r g e s and s c a t t e r i n g c r o s s s e c t i o n i n nm2
77 f o r i e n = 1 : l e n g t h ( lambda )
78 % s u r f a c e charge
81 % scattering cross section
84 end
85 end
86 %% T o t a l s c a t t e r i n g c r o s s s e c t i o n
87 s c a t o t = s c a 1 + s c a 2 ;
88
89 % Normalize s c a t t e r i n g f o r maximum s c a t t e r i n g c r o s s s e c t i o n
90 s c a t o t n o r m = s c a t o t . / max( s c a t o t ) ;
Lysanne Mol 48
D. Main script for evaluating the far-field effects of incident angle
1 %% Main s c r i p t f o r t e s t i n g t h e i n f l u e n c e o f t h e i n c i d e n t a n g l e f o r d i f f e r e n t
AuNP sizes .
2 % For each c o m b i n a t i o n o f p a r t i c l e s i z e and i n c i d e n t a n g l e , t h e s c a t t e r i n g c r o s s
3 % s e c t i o n i s c a l c u l a t e d f o r the given range o f wavelengths .
4 % Date : 02−06−2020
5 % Created by Lysanne Mol
7
8 film thickness = 50; % T h i c k n e s s o f g o l d l a y e r [nm]
9 a n g l e r a n g e = [ 1 0 30 50 7 0 ] ; % I n c i d e n t a n g l e o f incoming l i g h t [ rad ]
10 e n e i = l i n s p a c e ( 4 5 0 , 1 0 0 0 , 1 0 0 ) ; % Wavelengths o f l i g h t [nm]
11
12 d i a m e t e r r a n g e = [ 4 0 8 0 ] ; % AuNP s i z e s t h a t a r e s i m u l a t e d [nm]
13 s p a c i n g r a n g e = 2 . 0 ; % Gap s i z e s t h a t a r e s i m u l a t e d [nm]
14
15 %% D e f i n e d i e l e c t r i c f u n c t i o n s
16
17 % Order o f d i e l e c t r i c c o n s t a n t s : PBS ( 0 . 1 0 mM) , g o l d ( p a r t i c l e ) , PEG, SEM, g o l d
( l a y e r ) , titanium , b o r o s i l i c a t e g l a s s
18 e p s t a b = { e p s c o n s t ( 1 . 3 3 8 ˆ 2 ) , e p s t a b l e ( ' g o l d . dat ' ) , e p s c o n s t ( 1 . 3 7 ˆ 2 ) , e p s c o n s t
( 1 . 4 0 5 ˆ 2 ) , e p s t a b l e ( ' g o l d . dat ' ) , e p s t a b l e ( ' t i t a n i u m . dat ' ) , e p s c o n s t ( 1 . 5 1 ˆ 2 ) } ;
19
20 %% D e f a u l t o p t i o n s f o r l a y e r s t r u c t u r e
21
23
24 %% C r e a t e a r r a y s t o s a v e s c a t t e r i n g data
25 % The f i r s t and s e c o n d d i m e n s i o n s a v e t h e data f o r t h e d i f f e r e n t
26 % wave lengths , t h e t h i r d d i m e n s i o n s a v e s t h e data f o r each i n c i d e n t a n g l e
27 % and t h e f o u r t h d i m e n s i o n f o r each AuNP s i z e . Using smart i n d e x i n g , t h e
28 % s c a t t e r i n g c r o s s s e c t i o n f o r each c o m b i n a t i o n o f AuNP s i z e and gap s i z e
29 % can be found .
30
31 s c a 1 = NaN( l e n g t h ( e n e i ) , 1 , l e n g t h ( a n g l e r a n g e ) , l e n g t h ( d i a m e t e r r a n g e ) ) ;
32 s c a 2 = NaN( l e n g t h ( e n e i ) , 1 , l e n g t h ( a n g l e r a n g e ) , l e n g t h ( d i a m e t e r r a n g e ) ) ;
33
34 f o r i a = 1 : l e n g t h ( a n g l e r a n g e ) % l o o p f o r each i n c i d e n t a n g l e
35 f o r i d = 1 : l e n g t h ( d i a m e t e r r a n g e ) % l o o p f o r each p a r t i c l e s i z e
36 angle = angle range ( i s ) % current incident angle
37 diameter = diameter range ( id ) % c u r r e n t AuNP s i z e
38 %% D e f i n e l a y e r s t r u c t u r e
39 % z t a b i s a t a b l e which c o n t a i n s a l l t h e i n t e r f a c e s between t h e
40 % d i f f e r e n t d i e l e c t r i c c o n s t a n t s , s t a r t i n g a t t h e top .
41 z t a b = [−1 * ( d i a m e t e r /2 + s p a c i n g ) , −1 * ( d i a m e t e r /2 + s p a c i n g )− 0 . 9 ,
−1 * ( d i a m e t e r /2 + s p a c i n g )− 0 . 9 − 1 . 5 , −1 * ( d i a m e t e r /2 + s p a c i n g )
− 0.9 − 1.5 − film thickness , −1 * ( d i a m e t e r /2 + s p a c i n g )− 0 . 9 −
1.5 − film thickness − 5 ] ;
42
43 % d e f i n e the order of the l a y e r s
44 l a y e r = l a y e r s t r u c t u r e ( epstab , [ 1 , 3 , 4 , 5 , 6 , 7 ] , ztab , op ) ;
45
46 %% BEM o p t i o n s
47
49
50 %% D e f i n e incoming l i g h t
51
52 theta = angle * pi /180;
Lysanne Mol 49
53
58
63
65
68
69 % combine p a r t i c l e with l a y e r s t r u c t u r e and d i e l e c t r i c f u n c t i o n s
71
72 %% Compute Green f u n c t i o n s
73
81
83
86 % f o r each wavelength , t h e s u r f a c e c h a r g e and s c a t t e r i n g c r o s s
87 % section is calculated
88 for ien = 1 : length ( enei )
89 % Surface charge
90 s i g 1 = bem \ exc1 ( p , e n e i ( i e n ) ) ;
92 % Scattering cross section
93 s c a 1 ( i e n , : , i a , i d ) = abs ( exc1 . s c a ( s i g 1 ) ) ;
94 s c a 2 ( i e n , : , i a , i d ) = abs ( exc2 . s c a ( s i g 2 ) ) ;
95 end
96 end
97 end
98
99 %% T o t a l s c a t t e r i n g
100
101 s c a t o t = s c a 1 + s c a 2 ;
Lysanne Mol 50
E. Main script for evaluating the near-field effects of incident angle
1 %% S c r i p t f o r e v a l u a t i n g t h e near− f i e l d e f f e c t s o f v a r y i n g i n c i d e n t a n g l e
2 % For each c o m b i n a t i o n o f p a r t i c l e s i z e and i n c i d e n t a n g l e , t h e near− f i e l d and
maximum f i e l d enhancement
3 % i s c a l c u l a t e d f o r t h e g i v e n wavelength .
4 % Date : 02−06−2020
6
8 film thickness = 50;
9 a n g l e r a n g e = [ 1 0 30 50 7 0 ] ;
10 v e r t i c e s = 2 0 0 ;
11 e n e i = 7 6 5 ;
12
13 d i a m e t e r r a n g e = [ 4 0 8 0 ] ;
14 s p a c i n g = 2 ;
15
18
19 %% S e t up data s t o r a g e
22 f i e l d = NaN( 2 0 1 , 1 6 1 , l e n g t h ( a n g l e r a n g e ) , l e n g t h ( d i a m e t e r r a n g e ) ) ;
23
24 %% C a l c u l a t i o n s
25 f o r i a = 1 : l e n g t h ( a n g l e r a n g e )
26 for id = 1: length ( diameter range )
27 angle = angle range ( ia )
28 diameter = diameter range ( id )
30
31 % location of interface of substrate
32 z t a b = [−1 * ( d i a m e t e r /2 + s p a c i n g ) , −1 * ( d i a m e t e r /2 + s p a c i n g )− 0 . 9 , −1
* ( d i a m e t e r /2 + s p a c i n g )− 0 . 9 − 1 . 5 , −1 * ( d i a m e t e r /2 + s p a c i n g )− 0 . 9 −
1.5 − film thickness , −1 * ( d i a m e t e r /2 + s p a c i n g )− 0 . 9 − 1 . 5 −
film thickness − 5];
35
36 % TM mode , e x c i t a t i o n from above
40
45
46 % Create p a r t i c l e
47 p = t r i s p h e r e ( v e r t i c e s , diameter ) ;
49
50
53 pt = compoint ( p , [ x ( : ) , 0 * x ( : ) , z ( : ) ] , op ) ;
Lysanne Mol 51
56 g r e e n t a b = s e t ( g r e e n t a b , e n e i , op , ' w a i t b a r ' , 1 ) ;
58
61
62 % c a l c u l a t e s u r f a c e charge
63 s i g 1 = bem \ exc1 ( p , e n e i ) ;
64 s i g 2 = bem \ exc2 ( p , e n e i ) ;
65
66 % emission pattern
67 % angles
69
72
75
76 % computation o f e l e c t r i c f i e l d
78
80 e1 = emesh ( s i g 1 ) + emesh ( exc1 . f i e l d ( emesh . pt , e n e i ) ) ;
82
84 e e 1 = s q r t ( dot ( e1 , e1 , 3 ) ) ;
85 e e 2 = s q r t ( dot ( e2 , e2 , 3 ) ) ;
86 ee = ee1 + ee2 ;
87 f i e l d ( 1 : 2 0 1 , 1 : 1 6 1 , ia , id ) = ee ;
88 end
89 end
90
91 %% C a l c u l a t i n g maximum f i e l d enhancement f o r each i n c i d e n t a n g l e
92 %40 nm AuNPs
93 Enhance40 = NaN( l e n g t h ( a n g l e r a n g e ) ) ;
94 f o r k = 1 : l e n g t h ( a n g l e r a n g e )
95 Enhance40 ( k ) = max(max( f i e l d ( : , : , k , 1 ) ) ) ;
96 end
97
98 %80 nm AuNPs
99 Enhance80 = NaN( l e n g t h ( a n g l e r a n g e ) ) ;
100 f o r m = 1 : l e n g t h ( a n g l e r a n g e ) ;
101 Enhance80 (m) = max(max( f i e l d ( : , : , m, 2 ) ) ) ;
102 end
Lysanne Mol 52
F. Main script for evaluating the far-field effects of AuNP size and
gap size
1 %% Main s c r i p t f o r t e s t i n g t h e i n f l u e n c e o f gap s i z e and AuNP s i z e
2 % For each c o m b i n a t i o n o f p a r t i c l e s i z e and gap s i z e , t h e s c a t t e r i n g c r o s s
3 % s e c t i o n i s c a l c u l a t e d f o r the given range o f wavelengths .
4 % Date : 02−06−2020
7
8 film thickness = 50; % T h i c k n e s s o f g o l d l a y e r [nm]
9 t h e t a = 60 * p i / 1 8 0 ; % I n c i d e n t a n g l e o f incoming l i g h t [ rad ]
10 e n e i = l i n s p a c e ( 450 , 1000 , 100) ; % Wavelengths o f l i g h t [nm]
11
12 diameter range = [40 8 0 ] ; % AuNP s i z e s t h a t a r e s i m u l a t e d [nm]
13 spacing range = [1.0 1.5 2 . 0 ] ; % Gap s i z e s t h a t a r e s i m u l a t e d [nm]
14
15 %% D e f i n e d i e l e c t r i c f u n c t i o n s
16
17 % Order o f d i e l e c t r i c c o n s t a n t s : PBS ( 0 . 1 0 mM) , g o l d ( p a r t i c l e ) , PEG, SEM, g o l d
( l a y e r ) , titanium , b o r o s i l i c a t e g l a s s
19
20 %% D e f a u l t o p t i o n s f o r l a y e r s t r u c t u r e
21
23
24 %% C r e a t e a r r a y s t o s a v e s c a t t e r i n g data
25 % The f i r s t and s e c o n d d i m e n s i o n s a v e t h e data f o r t h e d i f f e r e n t
26 % wave lengths , t h e t h i r d d i m e n s i o n s a v e s t h e data f o r each gap s i z e and t h e
27 % f o u r t h d i m e n s i o n f o r each AuNP s i z e . Using smart i n d e x i n g , t h e s c a t t e r i n g
28 % c r o s s s e c t i o n f o r each c o m b i n a t i o n o f AuNP s i z e and gap s i z e can be found .
29
30 s c a 1 = NaN( l e n g t h ( e n e i ) , 1 , l e n g t h ( s p a c i n g r a n g e ) , l e n g t h ( d i a m e t e r r a n g e ) ) ;
31 s c a 2 = NaN( l e n g t h ( e n e i ) , 1 , l e n g t h ( s p a c i n g r a n g e ) , l e n g t h ( d i a m e t e r r a n g e ) ) ;
32
33 f o r i s = 1 : l e n g t h ( s p a c i n g r a n g e ) % l o o p f o r each gap s i z e
34 f o r i d = 1 : l e n g t h ( d i a m e t e r r a n g e ) % l o o p f o r each p a r t i c l e s i z e
35 spacing = spacing range ( i s ) % c u r r e n t gap s i z e
36 diameter = diameter range ( id ) % c u r r e n t AuNP s i z e
37 %% D e f i n e l a y e r s t r u c t u r e
38 % z t a b i s a t a b l e which c o n t a i n s a l l t h e i n t e r f a c e s between t h e
39 % d i f f e r e n t d i e l e c t r i c c o n s t a n t s , s t a r t i n g a t t h e top .
40 z t a b = [−1 * ( d i a m e t e r /2 + s p a c i n g ) , −1 * ( d i a m e t e r /2 + s p a c i n g )− 0 . 9 ,
−1 * ( d i a m e t e r /2 + s p a c i n g )− 0 . 9 − 1 . 5 , −1 * ( d i a m e t e r /2 + s p a c i n g )
− 0.9 − 1.5 − film thickness , −1 * ( d i a m e t e r /2 + s p a c i n g )− 0 . 9 −
1.5 − film thickness − 5 ] ;
41
42 % d e f i n e the order of the l a y e r s
44
45 %% BEM o p t i o n s
46
48
49 %% D e f i n e incoming l i g h t
50
Lysanne Mol 53
55
60
62
65
66 % combine p a r t i c l e with l a y e r s t r u c t u r e and d i e l e c t r i c f u n c t i o n s
68
69 %% Compute Green f u n c t i o n s
70
78
80
83 % f o r each wavelength , t h e s u r f a c e c h a r g e and s c a t t e r i n g c r o s s
84 % section is calculated
85 for ien = 1 : length ( enei )
86 % Surface charge
89 % Scattering cross section
90 s c a 1 ( i e n , : , i s , i d ) = abs ( exc1 . s c a ( s i g 1 ) ) ;
91 s c a 2 ( i e n , : , i s , i d ) = abs ( exc2 . s c a ( s i g 2 ) ) ;
92 end
93 end
94 end
95
96 %% T o t a l s c a t t e r i n g
97
98 s c a t o t = s c a 1 + s c a 2 ;
Lysanne Mol 54
G. Main script for evaluating the near-field effects of AuNP size and
gap size
1 %% S c r i p t f o r e v a l u a t i n g t h e near− f i e l d e f f e c t s o f v a r y i n g AuNP s i z e and gap
size
2 % For each c o m b i n a t i o n o f p a r t i c l e s i z e and gap s i z e , t h e near− f i e l d and maximum
f i e l d enhancement
3 % i s c a l c u l a t e d f o r t h e g i v e n wavelength .
4 % Date : 02−06−2020
6
8 film thickness = 50;
9 a n g l e r a n g e = [ 1 0 30 50 7 0 ] ;
10 v e r t i c e s = 2 0 0 ;
11 e n e i = 7 6 5 ;
12
13 d i a m e t e r r a n g e = [ 4 0 8 0 ] ;
14 s p a c i n g = [ 1 2 3 4 5 7 10 15 20 30 4 0 ] ;
15
18
19 %% S e t up data s t o r a g e
22 f i e l d = NaN( 2 0 1 , 1 6 1 , l e n g t h ( s p a c i n g r a n g e ) , l e n g t h ( d i a m e t e r r a n g e ) ) ;
23
24 %% C a l c u l a t i o n s
25 f o r i s = 1 : l e n g t h ( s p a c i n g r a n g e )
26 for id = 1: length ( diameter range )
27 spacing = spacing range ( i s )
28 diameter = diameter range ( id )
30
32 z t a b = [−1 * ( d i a m e t e r /2 + s p a c i n g ) , −1 * ( d i a m e t e r /2 + s p a c i n g )− 0 . 9 , −1
* ( d i a m e t e r /2 + s p a c i n g )− 0 . 9 − 1 . 5 , −1 * ( d i a m e t e r /2 + s p a c i n g )− 0 . 9 −
1.5 − film thickness , −1 * ( d i a m e t e r /2 + s p a c i n g )− 0 . 9 − 1 . 5 −
film thickness − 5];
35
36 % TM mode , e x c i t a t i o n from above
40
45
46 % Create p a r t i c l e
47 p = t r i s p h e r e ( v e r t i c e s , diameter ) ;
49
50
Lysanne Mol 55
53 pt = compoint ( p , [ x ( : ) , 0 * x ( : ) , z ( : ) ] , op ) ;
56 g r e e n t a b = s e t ( g r e e n t a b , e n e i , op , ' w a i t b a r ' , 1 ) ;
58
61
62 % c a l c u l a t e s u r f a c e charge
63 s i g 1 = bem \ exc1 ( p , e n e i ) ;
64 s i g 2 = bem \ exc2 ( p , e n e i ) ;
65
66 % emission pattern
67 % angles
69
72
75
76 % computation o f e l e c t r i c f i e l d
78
82
84 e e 1 = s q r t ( dot ( e1 , e1 , 3 ) ) ;
85 e e 2 = s q r t ( dot ( e2 , e2 , 3 ) ) ;
86 ee = ee1 + ee2 ;
87 f i e l d ( 1 : 2 0 1 , 1 : 1 6 1 , is , id ) = ee ;
88 end
89 end
90
91 %% C a l c u l a t i n g maximum f i e l d enhancement f o r each gap s i z e
92 %40 nm AuNPs
93 Enhance40 = NaN( l e n g t h ( s p a c i n g r a n g e ) ) ;
94 f o r k = 1 : l e n g t h ( s p a c i n g r a n g e )
95 Enhance40 ( k ) = max(max( f i e l d ( : , : , k , 1 ) ) ) ;
96 end
97
98 %80 nm AuNPs
99 Enhance80 = NaN( l e n g t h ( s p a c i n g r a n g e ) ) ;
100 f o r m = 1 : l e n g t h ( s p a c i n g r a n g e ) ;
101 Enhance80 (m) = max(max( f i e l d ( : , : , m, 2 ) ) ) ;
102 end
Lysanne Mol 56
H. Script for simulating the scattering intensity over time
1 %% S c r i p t t o e v a l u a t e s c a t t e r i n g o v e r time
2 % This s c r i p t i s made t o p r o c e s s t h e data from t h e Brownian Dynamics s c r i p t
3 % from Laurens S p o e l s t r a and combine t h i s with t h e look −up t a b l e s made by
4 % Lysanne Mol t o g e t s c a t t e r i n g −over−time r e s u l t s .
5 % Made by : Lysanne Mol and Laurens S p o e l s t r a
6 % Date : 10−06−2020
7
8 %% S e t t i n g s
9 clear all ; close all ; clc
10 SAM = ( 1 . 5 + 0 . 9 ) * 1 e −9; %h e i g h t o f SAM [m]
11 multifile = true ; %a n a l y s e m u l t i p l e f i l e s
12 R2 cut = 0.99; %c u t o f f f o r tau c u r v e f i t t i n g a l g o r i t h m
13 tfit init = 3E−5; %i n i t i a l g u e s s o f f i t t i n g l e n g t h
14
15 %% Load t h e data f i l e s
16 if multifile
17 [ f i l e s , path ] = u i g e t f i l e ( ' * . mat ' , ' M u l t i S e l e c t ' , ' on ' ) ; %p l e a s e f e t c h a l l
from same f o l d e r !
18 nfiles = numel ( f i l e s ) ; %c a l c u l a t e number o f f i l e s
19 elseif ˜multifile
20 [ f i l e s , path ] = u i g e t f i l e ( ' * . mat ' ) ;
21 n f i l e s = 1;
22 f i l e s = { f i l e s , f i l e s } ; %t r i c k t o be a b l e t o u s e same b l o c k o f code below
23 end
24 % 1 . 1 Prepare s t r u c t s t o s a v e t h e v a l u e s i n
25 fresults = struct ;
26 fparams = struct ;
27 filetemp = [];
28 restemp = [];
29
30 % 1 . 2 Read and s a v e i m p o r t a n t v a r i a b l e s
31 f o r i = 1 : n f i l e s
32 %each row i n t h e s e s t r u c t s r e p r e s e n t s a s e p a r a t e f i l e
33 f i l e t e m p = f u l l f i l e ( c h a r ( path ) , c h a r ( f i l e s ( i ) ) ) ;
34 restemp = l o a d ( f i l e t e m p ) ;
35 f r e s u l t s ( i ) . t s i m = restemp . r e s u l t s . t ;
36 f r e s u l t s ( i ) . zsim = restemp . r e s u l t s . z ;
37 f r e s u l t s ( i ) . r s i m = restemp . r e s u l t s . r ;
38 fparams ( i ) . Lbp = restemp . params . Lbp ;
39 fparams ( i ) . dBead = restemp . params . dBead ;
40 fparams ( i ) . tend = restemp . params . tend ;
41 fparams ( i ) . dt = restemp . params . dt ;
42 fparams ( i ) . s t o r e e v e r y = restemp . params . s t o r e e v e r y ;
43 f r e s u l t s ( i ) . Dsim = restemp . r e s u l t s . Dsim ;
44 f r e s u l t s ( i ) . mnDsim = mean ( f r e s u l t s ( i ) . Dsim ) ;
45 end
46
47
48 %% Tau c u r v e f i t t i n g
49 f o r i i = 1 : n f i l e s
50 % Making t h e f i l e s even l e n g t h e d f o r l a t e r u s e
51 i f rem ( l e n g t h ( f r e s u l t s ( i i ) . t s i m ) , 2 ) ˜= 0
52 f r e s u l t s ( i i ) . t s i m = f r e s u l t s ( i i ) . t s i m ( 2 : end ) ;
53 f r e s u l t s ( i i ) . zsim = f r e s u l t s ( i i ) . zsim ( 2 : end ) ;
54 f r e s u l t s ( i i ) . r s i m = f r e s u l t s ( i i ) . r s i m ( 2 : end ) ;
55 end
56 % C a l c u l a t i n g t h e a u t o c o r r e l a t i o n s o f r s i m and zsim
57 N = length ( f r e s u l t s ( i i ) . tsim ) ;
Lysanne Mol 57
58 fparams ( i i ) .N = N;
59 f r e s u l t s ( i i ) . l a g s = 0 : ( ( 2 * N−1) / 2 ) ; %we t a k e t h e p o s i t i v e a u t o c o r r e l a t i o n
only
60 f r e s u l t s ( i i ) . t l a g s = f r e s u l t s ( i i ) . l a g s . * ( f r e s u l t s ( i i ) . t s i m ( 3 )− f r e s u l t s ( i i ) .
tsim (2) ) ;
61 f r e s u l t s ( i i ) . c o r r r = x c o r r ( f r e s u l t s ( i i ) . rsim , ' c o e f f ' ) ;
62 f r e s u l t s ( i i ) . c o r r r = f r e s u l t s ( i i ) . c o r r r ( ( 2 * N−1) / 2 : ( 2 * N−1) ) ;
63 f r e s u l t s ( i i ) . c o r r z = x c o r r ( f r e s u l t s ( i i ) . zsim , ' c o e f f ' ) ;
64 f r e s u l t s ( i i ) . c o r r z = f r e s u l t s ( i i ) . c o r r z ( ( 2 * N−1) / 2 : ( 2 * N−1) ) ;
65 f r e s u l t s ( i i ) . lncorr r = log ( f r e s u l t s ( i i ) . corr r ) ;
66 f r e s u l t s ( i i ) . lncorr z = log ( f r e s u l t s ( i i ) . corr z ) ;
67
68 % Curve f i t t i n g
69 k = 1;
70 fresults ( i i ) . t f i t r (k) = t f i t i n i t ;
71 fresults ( i i ) . t f i t z (k) = t f i t i n i t ;
72 f r e s u l t s ( i i ) . R2r ( k ) = 0;
73 f r e s u l t s ( i i ) . R2z ( k ) = 0;
74 f r e s u l t s ( i i ) . taur (k) = NaN ;
75 f r e s u l t s ( i i ) . tauz ( k ) = NaN ;
76
77 % Fit rsim f i r s t
78 w h i l e f r e s u l t s ( i i ) . R2r ( k ) <= R2 cut
79 i c d t c o r r = f i n d ( f r e s u l t s ( i i ) . t s i m <= f r e s u l t s ( i i ) . t f i t r ( k ) , 1 , ' l a s t ' ) ;
80 fresults ( ii ) . tlags r fit = fresults ( i i ) . tlags (1: icd t corr ) ;
81 fresults ( i i ) . lncorr r fit = fresults ( i i ) . lncorr r (1: icd t corr ) ;
82 fl1 = fitlm ( fresults ( i i ) . tlags r fit , fresults ( i i ) . lncorr r fit , 'linear ' ,
'intercept ' , false ) ;
83 f r e s u l t s ( i i ) . t a u r ( k ) = abs ( 1 / f l 1 . C o e f f i c i e n t s . Estimate ) ;
84 f r e s u l t s ( i i ) . R2r ( k+1) = f l 1 . Rsquared . Ordinary ;
85 f r e s u l t s ( i i ) . t f i t r ( k+1) = f r e s u l t s ( i i ) . t f i t r ( k ) − 1E−7;
86 k = k+1;
87 end
88
89 % F i t zsim
90 k = 1;
91 w h i l e f r e s u l t s ( i i ) . R2z ( k ) <= R2 cut
92 i c d t c o r r = f i n d ( f r e s u l t s ( i i ) . t s i m <= f r e s u l t s ( i i ) . t f i t z ( k ) , 1 , ' l a s t ' ) ;
93 fresults ( ii ) . tlags z fit = fresults ( i i ) . tlags (1: icd t corr ) ;
94 fresults ( i i ) . lncorr z fit = fresults ( i i ) . lncorr z (1: icd t corr ) ;
95 fl1 = fitlm ( fresults ( i i ) . tlags z fit , fresults ( i i ) . lncorr z fit , 'linear ' ,
'intercept ' , false ) ;
96 f r e s u l t s ( i i ) . t a u z ( k ) = abs ( 1 / f l 1 . C o e f f i c i e n t s . Esti mate ) ;
97 f r e s u l t s ( i i ) . R2z ( k+1) = f l 1 . Rsquared . Ordinary ;
98 f r e s u l t s ( i i ) . t f i t z ( k+1) = f r e s u l t s ( i i ) . t f i t z ( k ) − 1E−7;
99 k = k+1;
100 end
101 end
102
103 %% P l o t tau v a l u e s i n a f i g u r e
104 f i g u r e
105 h o l d on
106 g r i d on ; g r i d minor ;
107 f o r i i = 1 : n f i l e s
108 s c a t t e r ( fparams ( i i ) . Lbp , f r e s u l t s ( i i ) . t a u r ( end ) . * 1 e6 , ' ˆ ' , ' MarkerEdgeColor ' , '#
EDB120 ' )
109 end
110
111 f i g u r e
112 h o l d on
Lysanne Mol 58
113 g r i d on ; g r i d minor ;
114 for i i = 1: n f i l e s
115 s c a t t e r ( fparams ( i i ) . Lbp , f r e s u l t s ( i i ) . t a u z ( end ) . * 1 e6 , ' ˆ ' , ' MarkerEdgeColor ' , '#
EDB120 ' )
116 end
117 figure
118 h o l d on
119 g r i d minor
120 for i i = 1: n f i l e s
121 s c a t t e r ( [ fparams ( i i ) . Lbp fparams ( i i ) . Lbp ] , f r e s u l t s ( i i ) . mnDsim . * 1 e18 , ' * ' )
122 end
123
124
125 %% Combination with BEM
126 %% Camera a v e r a g i n g and f i l t e r s e t t i n g s
127 s t o r e e v e r y 2 = 1 e −5; %r e s a m p l i n g f o r i n t e r p o l a t i o n
128 f i l t e r m i n = 580; %minimum o f f i l t e r i n nm
129 f i l t e r m a x = 620; %maximum o f f i l t e r i n nm
130 f p s = 1E4 ; %f r a m e r a t e o f t h e camera
131 avwin = 1/ f p s ; %window i n s e c o n d s
132 Winnum2 = round ( avwin / s t o r e e v e r y 2 ) ; %number o f v a l u e s i n a window
g i v e n by f p s
133
134 %% Data p r o c e s s i n g
135 % C a l c u l a t e G
136 f o r i i i = 1 : n f i l e s
137 G = ( f r e s u l t s ( i i i ) . zsim−SAM−fparams ( i i i ) . dBead / 2 ) . * 1 e9 ; %gap s i z e s i n nm,
W. r . t . PEG l a y e r
138 tsim = f r e s u l t s ( i i i ) . tsim ;
139 f o r i i = 1 : l e n g t h (G)
140 i f G( i i )<0
141 G( i i ) = 0 ;
142 end
143 end
144
145 % Ca lcu lat ing the s c a t t e r i n g
146 i f round ( fparams ( i i i ) . dBead * 1 e9 ) == 80
147 s c a = l o a d ( ' s c a 8 0 0 3 0 6 . mat ' ) ;
148 sca = sca . sca 80 ;
149 e l s e i f round ( fparams ( i i i ) . dBead * 1 e9 ) == 40
150 s c a = l o a d ( ' s c a 4 0 0 3 0 6 . mat ' ) ;
151 sca = sca . sca 40 ;
152 end
153 e n e i = l i n s p a c e ( 450 , 1000 , 100) ; %wavelength r a n g e i n
nm
154
155 % Resampling f o r i n t e r p o l a t i o n
156 i f rem ( l e n g t h (G) , 2 ) ˜= 0
157 G = G( 1 : end −1) ;
158 t s i m = t s i m ( 1 : end −1) ;
159 end
160 Winnum = round ( s t o r e e v e r y 2 / fparams ( i i i ) . s t o r e e v e r y ) ;
161 Nnum = round ( l e n g t h (G) /Winnum) ;
162 G = r e s h a p e (G, [ Winnum Nnum ] ) ;
163 G = mean (G) ;
164 t s i m = r e s h a p e ( tsim , [ Winnum Nnum ] ) ;
165 tsim = tsim ( 1 , : ) ;
166
167 % Interpolate
168 tic
Lysanne Mol 59
169 s c a t i m e = NaN( l e n g t h (G) , l e n g t h ( e n e i ) ) ; %C r e a t e a r r a y t o
s a v e s c a t t e r i n g data
170 f o r k = 1 : l e n g t h (G)
171 s c a t i m e ( k , : ) = i n t e r p 1 ( s c a .G, s c a . sca ,G( k ) ) ; %For each G, f i n d t h e
n e a r e s t G i n t h e t a b l e s c a 8 0 and f i n d t h e c o r r e s p o n d i n g s c a t t e r i n g
data
172 end
173 toc
174
175 % Apply f i l t e r
176 %Find t h e wavelength i n e n e i t h a t i s t h e c l o s e s t t o t h e minimum o f t h e
filter
177 [ wavelength min , i f m i n ] = min ( abs ( f i l t e r m i n − e n e i ) ) ;
178 %Find t h e wavelength i n e n e i t h a t i s t h e c l o s e s t t o t h e maximum o f t h e
filter
179 [ wavelength max , ifmax ] = min ( abs ( f i l t e r m a x − e n e i ) ) ;
180 %s c a f i l t e r g i v e s t h e s c a t t e r i n g f o r t h e d i f f e r e n t gap s i z e s f o r
181 %w a v e l e n g t h s w i t h i n t h e f i l t e r r a n g e
182 s c a f i l t e r = s c a t i m e ( : , i f m i n : ifmax ) ;
183
184 % Calculate scattering intensity
185 s t e p s z e n e i = e n e i ( 2 )−e n e i ( 1 ) ; %c o n s t a n t b e c a u s e produced by l i n s p a c e
186 f o r x = 1 : l e n g t h (G)
187 %C a l c u l a t e s c a t t e r i n g i n t e n s i t y f o r each gap s i z e w i t h i n f i l t e r r a n g e
188 s c a i n t e n s i t y (x) = s t e p s z e n e i * trapz ( s c a f i l t e r (x , : ) ) ;
189 end
190
191 f r e s u l t s ( i i i ) .G = G;
192 fresults ( i i i ) . sca intensity = sca intensity ;
193 f r e s u l t s ( i i i ) . tsim = tsim ;
194
195 % Apply camera
196 Nnum2 = round ( l e n g t h (G) /Winnum2) ;
197 s c a r e s h a p e d = r e s h a p e ( s c a i n t e n s i t y , [ Winnum2 Nnum2 ] ) ;
198 G reshaped = r e s h a p e (G, [ Winnum2 Nnum2 ] ) ;
199 s c a c a p = mean ( s c a r e s h a p e d ) ;
200 G cap = mean ( G reshaped ) ;
201 t s i m r e s h a p e d= r e s h a p e ( tsim , [ Winnum2 Nnum2 ] ) ;
202 tsim cap = tsim reshaped ( 1 , : ) ;
203
204 % Save data
205 f r e s u l t s ( i i i ) . G cap = G cap ;
206 f r e s u l t s ( i i i ) . sca cap = sca cap ;
207 f r e s u l t s ( i i i ) . tsim cap = tsim cap ;
208 f r e s u l t s ( i i i ) . mnSca = mean ( s c a c a p ) ;
209 end
210
211 %% P l o t gap s i z e o v e r time
212 f i g u r e
213 h o l d on
214 f o r i = 1 : n f i l e s
215 p l o t ( f r e s u l t s ( i ) . t s i m c a p . * 1 e3 , f r e s u l t s ( i ) . G cap , '−o ' , ' LineWidth ' , 2 )
216 end
217 l e g e n d ( ' 50 bp ' , ' 60 bp ' )
218 x l a b e l ( ' Time (ms) ' ) ; y l a b e l ( 'Gap s i z e (nm) ' )
219 xlim ( [ 0 1 ] )
220 t i t l e ( 'Gap s i z e o v e r time ' )
221 s e t ( gca , ' f o n t s i z e ' , 1 8 )
222
223 %% P l o t s c a t t e r i n g o v e r time
Lysanne Mol 60
224 figure
225 h o l d on
226 for i = 1: n f i l e s
227 p l o t ( f r e s u l t s ( i ) . t s i m c a p . * 1 e3 , f r e s u l t s ( i ) . s c a c a p , '−o ' , ' LineWidth ' , 2 )
228 y l a b e l ( ' S c a t t e r i n g i n t e n s i t y (nmˆ 3 ) ' )
229 end
230 y l i n e ( mean ( f r e s u l t s ( 1 ) . s c a c a p ) , '−−' , ' C o l o r ' , [ 0 0 . 4 4 7 0 0 . 7 4 1 0 ] , ' LineWidth ' , 2 ) ;
231 y l i n e ( mean ( f r e s u l t s ( 2 ) . s c a c a p ) , '−−' , ' C o l o r ' , [ 0 . 8 5 0 0 0 . 3 2 5 0 0 . 0 9 8 0 ] , ' LineWidth '
,2) ;
232 l e g e n d ( ' 50 bp ' , ' 60 bp ' )
233 x l a b e l ( ' Time (ms) ' ) ;
234 xlim ( [ 0 1 ] )
235 t i t l e ( ' S c a t t e r i n g o v e r time ' )
236 s e t ( gca , ' f o n t s i z e ' , 1 8 )
237
238 %% P l o t mean s c a t t e r i n g f o r d i f f e r e n t t e t h e r l e n g t h s
239 f i g u r e
240 h o l d on
241 f o r i = 1 : n f i l e s
242 s c a t t e r ( fparams ( i ) . Lbp , f r e s u l t s ( i ) . mnSca , ' MarkerEdgeColor ' , '#EDB120 ' , '
linewidth ' ,7)
243 end
244 x l a b e l ( ' Length o f dsDNA t e t h e r ( bp ) ' ) ; y l a b e l ( ' S c a t t e r i n g i n t e n s i t y (nm $ ˆ3 $ ) ' ) ;
245 t i t l e ( ' R e l a t i o n between t e t h e r l e n g t h and mean s c a t t e r i n g i n t e n s i t y − 40 nm bead
')
246 s e t ( gca , ' f o n t s i z e ' , 1 8 )
247
248 %% F i t l i n e a r r e l a t i o n between f i l e s
249 f o r i = 1 : n f i l e s
250 Lbpvec ( i ) = fparams ( i ) . Lbp ;
251 mnScavec ( i ) = f r e s u l t s ( i ) . mnSca ;
252 end
Lysanne Mol 61

Thesis Lysanne Mol 29 06 2020

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Summary

1 Introduction and Literature Overview 1

3 Influencing Factors of Surface Plasmon Resonance: Script Development 12

4 Influencing Factors of Surface Plasmon Resonance: Simulations 18

5 Scattering Fluctuation of a Particle-on-Film System over Time: Script Development 25

6 Scattering Fluctuation of a Particle-on-Film System over Time: Simulations 28

Introduction and Literature Overview

1.1 DNA hypermethylation

1.2 Microfluidic device for hmDNA detection

1.3 Research goals and expectations

2.1 Theory behind plasmon resonance

2.1.2 Excitation of localized surface plasmons

2.1.3 Damping of the oscillating effect

2.1.4 Dipole approximation

The dielectric function can in general be calculated by:

m (ω) + 2d = 0 (2.8)

2.1.5 Near-field and far-field effects of plasmon resonance

Cabs = Cext − Csca (2.12)

In case of the dipole approximation, a and b can be defined as:

2.1.6 Plasmon coupling

2.2 Current research on plasmon resonance

2.3 Conclusion on the theory behind plasmon resonance

Influencing Factors of Surface Plasmon

3.1 Specifications of the simulated system

3.2 Script development

3.3 Script validation

3.3.2 Validation using experimental data

3.4 Effect of different layers

3.5 Conclusions on script development

Influencing Factors of Surface Plasmon

4.1 Effect of incident angle

The change in near-field effects as a result of a

4.2 Effect of AuNP size

Figure 4.6 shows the local electric fields for

4.3 Effect of gap size

4.4 Conclusions on the influencing factors of surface plasmon reso-

5.1 Look-up table for varying gap sizes

5.2 Simulated camera settings

5.2.2 Filter influence

5.3 Script development

5.4 Conclusions on script development

6.1 Time-dependent scattering intensity for different tether lengths

6.1.1 Simulating 40nm AuNPs

6.1.2 Simulating 80nm AuNPs

6.1.3 Relation between tether length and mean scattering intensity

6.2 Influence of standard deviation of particle size

6.3 Red-green ratio for different tether lengths

6.4 Dynamic variance of RG-ratio

6.5 Conclusions on scattering fluctuation over time

Discussion and Recommendations

7.1 Discussion on simulated results

7.2 Suggestions for further experiments

7.2.1 Imaging techniques

7.2.2 Experiments to validate the effect of influencing factors

7.2.3 Experiments to validate the scattering fluctuation over time

7.3 Suggestions on further simulations

7.3.1 Further simulations on influencing factors

m (ω) + 2d = 0 (2.8)