Optical Properties of metallized DNA

Kammerlander, Nadine

Optical Properties of metallized DNA

Zusammenfassung

Inhaltsangabe:Introduction:
Modern technologies cannot be imagined without electronic devices, as e.g. the success stories of transistors and diodes illustrate. The performance of these electronic devices has tremendously increased during the last decades through a process known as downscaling: Minimizing structural units allows for extremely high densities of electronic modules. This entails an increase of speed as well as an enhanced performance. Todays photonic devices, however, are still rather large. The reason therefore can be found in the nature of light: Diffraction sets a lower limit for the scales of photonic devices and therefore prevents densely packed photonic components. The diffraction limit can be, however, overcome by so called Surface Plasmon Polaritons (SPPs): These are oscillations of the conducting electrons in a metallic nanoparticle induced by an incoming electromagnetic field with a suitable frequency. By this conversion of the optical mode to surface plasmons, the electromagnetic energy can be localized to regions of merely several tens of nanometers. This property is highly desirable for the construction of future optoelectronic devices: Chains of metallic nanoparticles, for example, are promising waveguides with lateral confinements below the diffraction limit.
DNA is a potential pattern material for constructing nanostructures as it can be synthesized in almost arbitrary lengths in the nanometer scale and also allows for modifications that make site-specific reactions possible. Its inherent scaling limit is determined by the distance of two neighbored basepairs: This length is only 0.34nm and therefore much below the diffraction limit. Moreover, it seems very appealing to exploit the self-recognition properties of DNA to build functional nanostructures by self-assembly processes. Among all possible template-directed material syntheses, metallization of DNA is particularly interesting for photonic devices as it makes a controlled growth of metal nanoparticles with defined size and shape along the DNA template possible. Furthermore the use of DNA as a pattern promises to achieve a defined arrangement of the particles relative to each other.
Experiments concerning metallization of DNA have been done for approximately a decade now. Most experiments, however, have concentrated on investigating the electrical conductance of the DNA. Absorption spectra have merely been recorded for verifying the existence of metallized DNA […]

Leseprobe

Inhaltsverzeichnis

Nadine Kammerlander

Optical Properties of metallized DNA

ISBN: 978-3-8366-2212-7

Herstellung: Diplomica® Verlag GmbH, Hamburg, 2009

Zugl. Technische Universität München, München, Deutschland, Diplomarbeit, 2007

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Abstract

Metallic nanoparticles have been studied intensively during the last decades because of

their intriguing optical properties: Due to collective oscillations of the conducting elec-

trons - the so called plasmonic oscillations - they absorb light in the visible spectrum. The

resonance frequency thereby sensitively depends on parameters such as the particle size

and shape as well as the dielectric constant of the medium. DNA exhibits outstanding

recognition properties and can be modified easily. Thus, template-directed material syn-

thesis along synthetic DNA is a promising route to grow nanoparticles of defined shape

and size and with defined interparticle-spacing.

In this study, two different methods are used to deposit silver on oligonucleotides of

different lengths, ranging from 23 to 96 basepairs, in order to synthesize metallic nanorods

of controlled aspect ratios. The first method involves the specific labeling of nucleotides

with aldehyde groups, followed by exposure to a Tollens reagent and a developer. The

second method relies on the photoinduced deposition of silver onto unmodified DNA

samples. Several preparation parameters such as the DNA sequence, buffer salt type,

silver concentration and UV illumination time are varied systematically.

The metallized DNA molecules are characterized concerning their optical and structural

properties. Absorption spectra show plasmon peaks around 420nm. Peak positions, inten-

sities and bandwidths are analyzed. Dynamic Light Scattering studies in solution provide

information about the particle sizes as well as their structural asymmetry. Both opti-

cal techniques are used to observe the temporal evolution of the nanoparticle growth in

the Tollens metallization process. Structural information is inferred from Atomic Force

Microscopy; for that purpose, the particles are deposited on single-crystalline silicon sub-

strates.

Zusammenfassung

Metallische Nanopartikel werden seit einigen Jahrzehnten intensiv erforscht, unter an-

derem, weil sie sich durch besondere optische Eigenschaften, sogennante Plasmonenreso-

nanzen, auszeichnen: Bei Einfall von Licht im sichtbaren Bereich zeigen die Teilchen starke

Absorption, welche durch kollektive Schwingungen der Leitungsbandelektronen bedingt

ist. Die Wellenl¨

ange dieser Plasmonenresonanz ist abh¨

angig von diversen Parametern,

darunter Eigenschaften der Nanopartikel, wie Gr¨

oße und Form, sowie die dielektrischen

Eigenschaften des umgebenden Mediums. Der Einsatz von synthetischer DNS als Vorlage,

entlang derer Nanopartikel wachsen, ist sehr vielversprechend: Aufgrund ihrer Selbsterken-

nungseigenschaften sowie der vorhandenen M¨

oglichkeit, die DNS zu modifizieren, k¨

onnen

zum einen die Gr¨

oße und Geometrie der Teilchen vorgegeben werden. Zum anderen k¨

onnen

die Nanopartikel in einem definierten Abstand zueinander erzeugt werden.

In dieser Arbeit werden zwei verschiedene Methoden angewendet um Silber an der DNS

anzulagern. Um metallische Nanost¨

abchen mit kontrollierten Achsenverh¨

altnissen herzu-

stellen, werden verschiedene DNS-L¨

angen im Bereich von 23 bis 96 Basenpaaren verwen-

det. F¨

ur die erste Metallisierungsmethode werden an bestimmte Nukleotide Aldehydgrup-

pen angebunden. Anschließend wird zur L¨

osung ein Tollensreagenz sowie eine Entwick-

lerl¨

osung zugegeben. Die zweite Metallisierungsmethode besteht in einer UV-induzierten

Reduktion von Silberionen an unmodifizierter DNS. Bei der Synthese werden verschiedene

Parameter, darunter die DNS-Sequenz, die Pufferl¨

osung, die zugegebene Silberkonzentra-

tion sowie die Bestrahlungsdauer systematisch variiert.

Die in L¨

osung metallisierte DNS wird anschließend bez¨

uglich ihrer optischen und struk-

turellen Eigenschaften charakterisiert: Absorptionsspektren zeigen die f¨

ur Silbernanopar-

tikel typischen Maxima bei 420nm, die durch die Plasmonenoszillationen hervorgerufen

werden. F¨

ur die verschiedenen Proben werden die Position des Maximums, seine In-

tensit¨

at sowie die Halbwertsbreite bestimmt. Messungen der dynamischen Lichtstreuung

geben Aufschluss ¨

uber die Teilchengr¨

oßen in der L¨

osung und ihre H¨

aufigkeit. Polarisierte

Lichtstreuungsmessungen geben den Grad der Asymmetrie f¨

ur die Teilchen an. Beide

optischen Charakterisierungsmethoden werden verwendet um die zeitliche Entwicklung

des Teilchenwachstums bei der Tollens-Metallisierung zu beobachten. Aus Untersuchun-

gen mit einem Rasterkraftmikroskop lassen sich Informationen ¨

uber die Struktur der

metallisierten DNS-Molek¨

ule ableiten; daf¨

ur werden die Teilchen auf ein einkristallines

Siliziumsubstrat aufgebracht.

Contents

Motivation

Fundamentals

2.1

Metallic Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.2

Optical Properties of Nanospheres . . . . . . . . . . . . . . . . . . . . . . .

2.2.1

Extinction of Light . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.3

Optical Properties of Nanorods . . . . . . . . . . . . . . . . . . . . . . . .

2.3.1

Electrostatic Calculations

. . . . . . . . . . . . . . . . . . . . . . .

2.4

Deoxyribonucleic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.4.1

Composition and Properties . . . . . . . . . . . . . . . . . . . . . .

2.4.2

Metallized DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Synthesis of Metallized DNA

3.1

Hybridization of DNA

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.2

Requirements for Metallization

. . . . . . . . . . . . . . . . . . . . . . . .

3.3

Photochemical Metallization . . . . . . . . . . . . . . . . . . . . . . . . . .

3.4

Tollens Metallization of Aldehyde-Modified DNA

. . . . . . . . . . . . . .

3.5

Materials Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Absorption Spectrometry

4.1

Principle of Absorption Spectrometry . . . . . . . . . . . . . . . . . . . . .

4.2

Absorption Measurements of UV-Metallized Samples . . . . . . . . . . . .

4.2.1

Influence of pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

iii

CONTENTS

4.2.2

DNA Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2.3

Silver Amount . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2.4

Irradiation Time

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2.5

DNA Length

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2.6

Reference Samples

. . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2.7

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.3

Absorption Measurements of Tollens-Metallized Samples . . . . . . . . . .

4.3.1

Absorption Measurements of Tollens-Metallized dsDNA . . . . . . .

4.3.2

Absorption Measurements of Tollens-Metallized ssDNA . . . . . . .

4.3.3

Development Solution

. . . . . . . . . . . . . . . . . . . . . . . . .

4.3.4

Data Analysis and Discussion . . . . . . . . . . . . . . . . . . . . .

Dynamic Light Scattering

5.1

Principle of Dynamic Light Scattering

. . . . . . . . . . . . . . . . . . . .

5.1.1

Measurement Technique . . . . . . . . . . . . . . . . . . . . . . . .

5.1.2

Fundamentals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.1.3

Polarized Dynamic Light Scattering . . . . . . . . . . . . . . . . . .

5.1.4

Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2

Unpolarized Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2.1

Experimental Results . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2.2

CONTIN Analysis

. . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2.3

Temporal Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2.4

Reference Samples

. . . . . . . . . . . . . . . . . . . . . . . . . . .

5.3

Polarized Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.3.1

Simulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.3.2

Experimental Results . . . . . . . . . . . . . . . . . . . . . . . . . .

Stabilization of the Nanoparticles

6.1

Fundamentals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

CONTENTS

6.2

Experimental Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Nanorings

Atomic Force Microscopy

8.1

Fundamentals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8.2

Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8.2.1

Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8.2.2

Functionalization of Silicon Wafers . . . . . . . . . . . . . . . . . .

8.3

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8.3.1

Ultrasonication . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8.3.2

MPTMS-Silanization . . . . . . . . . . . . . . . . . . . . . . . . . .

8.3.3

Spin-Coating

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Conclusion

10 Outlook

A DNA Sequences

B Reference Samples

B.1 Absorption Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B.2 DLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B.3 AFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C Parameters of MPTMS-Silanization

D Acknowledgments

100

Chapter 1

Motivation

Modern technologies cannot be imagined without electronic devices, as e.g. the success

stories of transistors and diodes illustrate. The performance of these electronic devices has

tremendously increased during the last decades through a process known as downscaling:

Minimizing structural units allows for extremely high densities of electronic modules. This

entails an increase of speed as well as an enhanced performance. Today's photonic devices,

however, are still rather large. The reason therefore can be found in the nature of light:

Diffraction sets a lower limit for the scales of photonic devices and therefore prevents

densely packed photonic components. The diffraction limit can be, however, overcome by

so called 'Surface Plasmon Polaritons' (SPPs): These are oscillations of the conducting

electrons in a metallic nanoparticle induced by an incoming electromagnetic field with a

suitable frequency. By this conversion of the optical mode to surface plasmons, the elec-

tromagnetic energy can be localized to regions of merely several tens of nanometers. This

property is highly desirable for the construction of future optoelectronic devices: Chains

of metallic nanoparticles, for example, are promising waveguides with lateral confinements

below the diffraction limit.

DNA is a potential pattern material for constructing nanostructures as it can be synthe-

sized in almost arbitrary lengths in the nanometer scale and also allows for modifications

that make site-specific reactions possible. Its inherent scaling limit is determined by the

distance of two neighbored basepairs: This length is only 0.34nm and therefore much be-

low the diffraction limit. Moreover, it seems very appealing to exploit the self-recognition

properties of DNA to build functional nanostructures by self-assembly processes. Among

all possible template-directed material syntheses, metallization of DNA is particularly in-

teresting for photonic devices as it makes a controlled growth of metal nanoparticles with

defined size and shape along the DNA template possible. Furthermore the use of DNA

as a pattern promises to achieve a defined arrangement of the particles relative to each

other.

Experiments concerning metallization of DNA have been done for approximately a decade

now. Most experiments, however, have concentrated on investigating the electrical con-

ductance of the DNA. Absorption spectra have merely been recorded for verifying the

CHAPTER 1. MOTIVATION

existence of metallized DNA in the solution. The optical properties of metallic nanopar-

ticles grown on DNA templates have remained almost completely unexplored up to now.

Thus it is the main topic of this thesis to investigate the optical properties of metal-

lized DNA structures, i.e. their absorption as well as scattering characteristics. Therefore

two different metallization methods are applied and the resulting metallized particles are

compared. Varying synthesis parameters, such as concentrations of the reagent or the re-

action time systematically and analyzing the optical data, provides an understanding on

what variables the metallization depends on. Changes in optical properties are related to

changes in the size or structure of the nanoparticles. This information is extracted from po-

larized Dynamic Light Scattering experiments as well as Atomic Force Microscopy. More-

over Dynamic Light Scattering allows for direct monitoring of the nanoparticle growth;

thereby the particle sizes as well as intensities are observed. These results are necessary

to understand the DNA metallization process in detail and to be able to optimize the

synthesis conditions on the way to a defined growth of metallic nanoparticles. Using var-

ious seed densities for site-specific DNA metallization and analyzing the resulting optical

properties is a fist step to a defined arrangement of nanoparticles.

This thesis is structured as follows: After a brief introduction into theory about the prop-

erties of metallic nanoparticles and about DNA in chapter 2, chapter 3 explains the

two different methods of metallization that are used for synthesis. Chapter 4 addresses

absorption spectrometry: After a short description of the working principle, absorption

spectra of metallized DNA samples are shown for numerous varied parameters and ana-

lyzed. The results are finally discussed and compared with two different theories about

nanoparticle absorption. Chapter 5 is about Dynamic Light Scattering: In a theoretical

part, simulations are done for a later comparison with experimental data. The second half

of the chapter presents first results for the various metallized DNA samples. In chapter 6

the necessity for stabilizers and their working principle is explained before some experi-

mental data is shown. Chapter 6 handles with metallization of ring-like DNA structures.

In chapter 8 micrographs recorded with an Atomic Force Microscope are presented and

analyzed.Afterward the obtained results of this thesis are concluded and an outlook for

future investigations is given.

Chapter 2

Fundamentals

2.1

Metallic Nanoparticles

Metallic nanoparticles have been used for more than a millennium, e.g. gold nanoparticles

were utilized for staining glass. 150 years ago Lord Faraday was one of the first to do

scientific work about metallic colloidal solutions. He recognized that the color of the

colloids originates from the small size of the particles.

Nowadays it is possible to produce nanoparticles in defined sizes and to a certain ex-

tent also with defined shapes. Important experiments have been done by Link, El Sayed

[LES99], and J. J. Mock et al. [MBS

02]. Transmission electron microscopy (TEM) im-

ages of pentagon- and triangle-shaped nanoparticles produced by Mock are shown in fig.

2.1.

Figure 2.1: TEM images of nanoparticles with a pentagon and a triangle shape, respectively.

This work was done by Mock in 2002, [MBS

02]

Production processes range from wet chemical synthesis to e-beam lithography. Whereas

the first method provides for highly monocrystalline particles, the latter allows for an

defined arrangement of the particles on a surface.

Fields of application are widely spread and include medicine as well as materials science.

Metallic nanoparticles are promising nanosensors due to their optical properties, the so

called 'surface plasmons', which will be explained later in 2.2.

As the name already tells, nanoparticles have sizes between a few and hundreds of nanome-

ters. The clusters typically consist of several up to thousands of atoms and differ a lot in

CHAPTER 2. FUNDAMENTALS

their properties from the bulk material.

One reason are surface effects, which become much more important as a larger percentage

of atoms is on the surface. For very small particles, e.g. silver particles with a diameter

smaller than about 52nm [SGHURS

05] the mean free path of the electrons is limited

mainly by collision with the surface. Quantum effects due to the confinement of space,

however, do not play an important role for the nanoparticles regarded here: The splitting

of energy levels can be approximated as E =

with the Fermi-energy E

=5.49eV

[AN05] and N the number of levels in the band [KV83]. To see quantum effects, the energy

splitting must be larger than the thermal energy k

T , 25meV at room temperature. This

is only fulfilled for clusters containing less than approximately 200 atoms.

2.2

Optical Properties of Nanospheres

As mentioned above metallic nanoparticles are characterized by particular optical prop-

erties. In the following an overview about the underlying physical fundamentals is given:

2.2.1

Extinction of Light

When light transverses a medium other than vacuum, dipoles are induced due to the

electromagnetic field. The nature of these dipoles depends on the medium and can be

either molecular, atomic or the displacement of the free electron gas in a conductor. The

'polarizability' indicates the dipole strength in dependence of the incoming field. It is

a frequency dependent material constant, that determines the response strength of the

material to an incoming field.

The induced dipoles themselves emit so called 'secondary radiation' in the toroidal radi-

ation pattern that is typical for dipoles. On a macroscopic scale, this secondary radiation

appears as scattered light. It is also possible that the medium releases the energy taken up

from the electromagnetic wave in a non-radiant way; this is absorption. Hence absorption

and scattering are not independent of each other. The sum of absorption and scattering

is called 'extinction'.

In electrodynamics all optical properties are characterized by the complex dielectric func-

tion , that depends on the medium as well as the frequency of the incoming field. It

describes Starting from this function, one can calculate further important physical values

such as the refractive index, the transmission or the absorption.

Scattering of Light

As mentioned before, the induced dipoles in the medium act as secondary scatterers for

an incoming electromagnetic wave. The interference of this scattered light determines the

2.2. OPTICAL PROPERTIES OF NANOSPHERES

Figure 2.2: The real and imaginary part of the dielectric function of bulk silver according to

[JC72]

angle-dependence of the outcoming light. Very small particles with a radius smaller than

/10 scatter unpolarized light isotropically as all the secondary waves are approximately

in phase. The intensity of the scattered light can be approximated as I

, the so called

Rayleigh approximation [BH83]. Huge particles larger than roughly 10, however, scatter

mainly in the forward direction. The scattered intensity is inversely proportional to the

scattering angle.

Absorption of Light by Plasmonic Oscillations

The Rayleigh formula in the previous subsection has already shown that scattering in-

creases with the particle size. For small nanoparticles absorption therefore predominates.

Absorption of light can be traced back to atomic or molecular transitions or to collective

phenomena. One of these - the plasmonic oscillations - is described in more detail as it is

important for nanoparticle analysis.

Plasmonic Oscillations

When the free electrons of a metallic conduction band are moved away from their equi-

librium position, the electromagnetic attraction between the negatively charged electrons

and the positively charged atomic cores counteracts the external force of the electro-

magnetic wave. This is the situation of a damped, driven harmonic oscillator. When the

external frequency matches the eigenfrequency of the electron system, the amplitude of

the electron movement increases, the electrons undergo so called plasmonic oscillations

and energy is absorbed. In general there are several methods for releasing the energy, such

as single electron excitation or heating up.

In bulk material, the dielectric function according to the Drude theory is () = 1-

-i

This term includes the damping constant =

with v

the Fermi velocity and l

the free

CHAPTER 2. FUNDAMENTALS

Figure 2.3: An incoming electromagnetic wave, whose electric field vectors are indicated with

black arrows, moves the electronic cloud of the metallic spheres out of equilibrium. The positively

charged atomic cores stay at their original position and therefore an attractive Coulomb force

acts on the electronic cloud. This induces oscillations. When the frequency of the incoming light

matches the eigenfrequency of the particles, light can be absorbed. This can be seen as a so

called plasmon peak in the absorption spectrum.

path length [AN05].

N e

is the plasmon frequency, that occurs at a wavelength

of 138nm for silver. Light with a wavelength longer than the plasmon peak wavelength is

reflected which leads the shiny glance that is typical for metals. These volume plasmon

waves are - like all oscillations in free gases - uniquely longitudinal waves and therefore

they cannot be excited by light, which is merely transverse. Thus, although plasmonic

oscillations are an important optical property of the bulk material, no incoming light can

be absorbed by exciting these plasmons. The dielectric function of bulk silver, determining

its optical properties, can be seen in fig. 2.2. Despite the plasmon wavelength, which

separates the region of reflection and transmission/absorption, there is also an important

feature at 318nm: At this wavelength the interband transitions set in.

Surface Plasmons

For surface plasmons the influence of the induced dipoles, that are located on the surface

of the particle and undergo oscillations, is important. Reflections of the electron cloud

on the surface change the boundary conditions of the problem: Shear forces induced by

such reflections entail a transverse component of the plasmon oscillation. Thus the surface

plasmons can, in contrast to the volume plasmons, indeed, be excited by light. For bulk

material, surface plasmons are negligible due to the small percentage of surface atoms. But

they gain importance with decreasing medium size and dominate the optical properties

of metallic nanoparticles. Fig. 2.3 shows schematically how plasmonic oscillations are

induced in a small metallic sphere.

2.2. OPTICAL PROPERTIES OF NANOSPHERES

The task to calculate the absorption of metallic nanoparticles is an electromagnetic one

and can therefore be done by solving the Maxwell equations in matter. Although this

is possible in general, there are indeed only few cases were exact solutions are known.

Mie solved the problem for spheres by expanding the incoming, internal and scattered

electromagnetic waves in an infinite series of vector spherical harmonics. These spherical

harmonics for the electric and the magnetic field are set in the Maxwell equations. The

boundary conditions determine the scattering coefficients for the specific problem. For

particles, not smaller than about 15nm, the size-independent dielectric function of the

bulk material can be used. A detailed derivation of the Mie theory is given by Bohren

and Huffman [BH83].

Figure 2.4: Plasmon peaks for commercial silver nanospheres with pM concentrations measured

with absorption spectroscopy. The spectra show that with increasing size, the peak shifts to the

red and becomes broader. The graph for r=40nm shows a quadrupole mode at 350nm.

Electrostatic Approximation

For small spherical particles the electrostatic approximation (also known as 'quasistatic

approximation') can be applied. The extinction cross section is thereby expressed by

dip

ext

3/2

()

(

() + 2

)

()

with R radius of the particle,

dielectric constant of the medium,

and

imaginary

and real part of the frequency dependent dielectric function of the nanoparticle respec-

tively. This electrostatic approximation estimates the position of the plasmon peak. It

shows that the intensity of the peak increases by the power of three with the particle

radius (i.e. it is proportional to the particle volume). The peak position is determined by

CHAPTER 2. FUNDAMENTALS

the zero of the denominator, i.e. by the equation

() = -2

. Thus the approximation

only depends on the material and the environment, but neglects the influence of size on

the exact peak position. The electrostatic approximation only accounts for the first terms

in the series of harmonics, the electrical dipole term. For particles with a radius larger

than 40nm, the quadrupole term is not negligible any more. An additional peak at a

higher energy occurs.

Although Mie theory is only valid for spherical particles and neglects any interaction

among the individual particles, it provides an understanding about what conditions the

plasmon peak position and shape depends on: The dielectric functions of the particle and

the medium are very important. There is also a dependence on the size: For small particles

the dielectric function itself is size-dependent. Theories about the effect of this intrinsic

feature differ. Larger particles show a red-shift with increasing size due to retardation

effects. Due to various damping-mechanisms, the peak width also depends on the particle

size. The width as well as the asymmetry of the peak are also influenced by the particle size

distribution when considering an ensemble of particles. Extinction spectra of commercially

available silver nanospheres provided by BBI/Tedpella Inc.

, Redding, CA is shown in fig.

2.4.

2.3

Optical Properties of Nanorods

Theory presented so far applies only to spherical particles. For non-spherical particles the

position as well as the number of absorption peaks in the spectrum changes. Ellipsoids

are the simplest case of non-spherical particles and there exists an extension of the Mie

theory to treat them. For ellipsoidal particles two cases can be imagined: Depending on the

relative orientation of the incoming field and the particle, a displacement of the electrons

along the short or the long axis of the particle is induced. In the first case one speaks of

'transverse plasmons', in the second case one speaks of 'longitudinal plasmons', its energy

is lower due to a longer wavelength. Fig. 2.5 schematically shows such a metallic nanorod

with the length L and the diameter d. For a collection of randomly orientated ellipsoids

two absorption peaks are expected in the spectrum corresponding to the longitudinal and

transversal plasmonic oscillation, respectively.

Figure 2.5: Schematic of a metallic nanorod with length L and diameter d.

Ag nanospheres were produced by reduction of silver citrate and therefore have a negative surface

charge. Depending on their size, their concentration lies between 20pM and 1.2nM.

2.3. OPTICAL PROPERTIES OF NANORODS

2.3.1

Electrostatic Calculations

To get a first idea about the plasmon peak positions of silver nanorods, I have done an

electrostatic calculation. This was based on Mie's electrostatic approximation for pro-

late spheroids [BH83]. For an electromagnetic field parallel to the principal axis i, the

absorption coefficient is thereby given as

() =

() -

+ [ () -

whereas

is the dielectric constant of the vacuum,

is the dielectric constant of the

embedding medium, () is the (complex) dielectric function of silver [JC72] and V is

the volume of the nanoparticles. P

are the geometrical depolarization factors, which are

given by the nanoparticle shape. Details can be found in [BH83]. They follow the sum

rule

= 1. For prolate ellipsoids the geometrical factor for one of the (identical) short

axes is

1 - e

-1 +

1 + e

1 - e

with the eccentricity

= 1 -

whereby a is the long and b is the short axis of the ellipsoid.

For

was set to 1.77 which equals the dielectric constant of pure water in the visible

spectrum

For randomly orientated ellipsoids only the average cross sections for absorption and

scattering, that are independent of the polarization of the incoming light, are of interest

[BH83]:

abs

= kIm

sca

These cross sections were calculated and are shown in fig. 2.6. The formulas already show

that maxima occur when the denominator of the absorption coefficient of any principal

axis becomes zero.

According to the calculation two peaks appear, that shift relatively to each other. This

shift, that is more pronounced for the longitudinal plasmon, is also visualized in fig. 2.7.

The value

= 80 that is frequently quoted for water is only valid for low frequencies. At about

10GHz the dielectric constant starts to decrease, as it is shown for example in [Fli00]

CHAPTER 2. FUNDAMENTALS

Figure 2.6: Electrostatic calculation of the plasmonic absorption of ellipsoids. Two peaks are

visible whereby the long-wavelength peak shifts to the red with increasing axis ratio and becomes

more intense.

Figure 2.7: According to electrostatic calculations the plasmon peaks are centered around 400nm.

The long-wavelength peak significantly shifts to the red with increasing non-sphericity whereas

the short-wavelength peak undergoes only a slight shift to the blue.

2.4. DEOXYRIBONUCLEIC ACID

2.4

Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) is not only important for biological matters but can be also

used for template-directed material synthesis. Thereby DNA is chosen as a pattern as it

can be easily synthesized with arbitrary length and sequence by solid state synthesis and

can even build up complex networks.

2.4.1

Composition and Properties

DNA is composed of a negatively charged phosphate backbone, pentose sugar molecules

as well as the four bases adenine (A), guanine (G), cytosine (C) and thymine (T) whose

sequence determine the code of the DNA. Natural DNA occurs in the so called 'B-form',

a right-handed rotated double helix with a base-to-base distance along the axis of 3.4nm.

For undergoing hybridization, i.e. forming a double helix, the bases have to match: A

and T are complementary bases, which can be bound via two hydrogen bonds. Another

complementary base pair is G and C, whose bond strength is slightly stronger due to

three hydrogen bonds. A schematical configuration of DNA is shown in fig. 2.8.

Figure 2.8: Configuration of double stranded DNA consisting of a phosphate backbone, sugars

and bases [Wik06]

Because of this hybridization, that takes place under certain conditions discussed in

[Man78], DNA possesses self-assembly properties that can be exploited for many ap-

plications in nanotechnology.

CHAPTER 2. FUNDAMENTALS

2.4.2

Metallized DNA

One possible application in nanotechnology is the growth of metallic nanoparticles on a

DNA pattern. First DNA metallization experiments were done by the group of Braun et

Keren [BESBY98]. They started coating DNA with metals by several chemical reactions

to provide for conductive nanowires. According to Eichhorn et al. [ES68] silver ions, albeit

they prefer binding to the bases instead of the backbone, do not unwind the double helix

and therefore do not destabilize the DNA. Various methods and metals (Pt, Ag, ...) have

been used by many groups working in this field. Richter presents a good summary about

experiments and results in this research area in his review paper [Ric03]. Most experi-

ments concentrate on µm-long DNA, e.g. -phage-DNA

, and result in several, randomly

distributed metal nanoclusters on the DNA, while homogeneous metal deposition in the

surrounding medium is suppressed. To yield a complete cover of the DNA, as intended by

Braun et al., an additional metallization step is necessary. This results in a wire diameter

of roughly 50nm that is 25 times larger than the diameter of bare double-stranded DNA.

To avoid a random placement of the nanoclusters, Braun invented a method to prevent

specific DNA parts from being metallized by incorporating RecA enzymes [KB04].

Silver clusters on DNA are typically in the nanometer size and therefore exhibit the

optical properties described above in 2.2 and 2.3. These optical properties and their de-

pendence on various synthesis as well as DNA parameters are researched in this work.

Moreover a method is applied to metallize only labeled parts of the DNA and the growth

of nanoclusters on short oligonucleotides is investigated.

DNA of the bacteriophage '-phage' with a length of approximately 50.000 basepairs

Chapter 3

Synthesis of Metallized DNA

In this chapter the metallization processes are explained in detail. Moreover the challenges

in synthesis are discussed.

The metallized nanoparticles are in general produced as colloids. Colloidal solutions can

be classified between real solutions and suspensions, meaning they consist of small clusters

dispersed in a solution: In contrast to suspensions, the clusters will not sediment after a

certain time as the buoyancy compensates for gravity. It is important to avoid aggregation

of the clusters caused by attractive van der Waals forces; this aspect is discussed in detail

in chapter 6.

3.1

Hybridization of DNA

To synthesize metallic rods on DNA instead of nanoparticles with arbitrary shape, it is

necessary to use double stranded DNA. The reason for this is, that the persistence length

of double-stranded DNA is roughly 50nm, whereas that of single stranded DNA is only

approximately 1nm, depending on the buffer solution [MVH99], [SCB96]. To achieve hy-

bridization of complementary DNA single strands, one has to provide a sufficient amount

of counter ions, such as sodium ions, in solution [Man78]. Furthermore, a moderate pH

value is necessary. Hybridization experiments were conducted in different buffer solutions

at room temperature.

The success of hybridization can be detected by measuring the absorption at 260nm: When

the DNA hybridizes, the light absorption of the DNA bases at 260nm decreases. This

can be attributed to Coulombic interactions of the stacked bases in the double stranded

DNA: The dipole transitions moments of the bases interact via Coulombic forces. The

outcome of this interaction depends on the relative orientation of the bases. By forming

a double helix secondary structure the dipole transition moments change from random

distribution to a parallel orientation as schematically shown in fig. 3.1. The square of the

The persistence length is a mechanical quantity that gives the stiffness of a of a polymer[Wik07]

CHAPTER 3. SYNTHESIS OF METALLIZED DNA

Figure 3.1: Principle of hypochromism for DNA: During hybridization the random distribution

of the DNA bases changes to a parallel alignment. The absorption of the molecule, which is

dependent on the relative orientation of the bases, thereby changes.

Figure 3.2: Absorption of 2µM of complementary 48mer DNA strands in 10mM Tris buffer

before and after hybridization: One spectrum is taken 10min after mixing the complementary

strands. The second spectrum is taken 35min later. A decrease in the absorption at 260nm can

be seen. This is attributed to the hypochromism effect and indicates hybridization.

dipole transition moment is directly proportional to the absorption of light. Therefore the

absorption intensity changes during hybridization. In 1960 Tinoco was the first to explain

this effect and to calculate it by perturbation theory calculations [Tin60].

For the concentrations (1µM) and lengths (ca. 10 up to 30nm) of DNA used for this work,

I typically observed changes between 5% and 15% in the absorption spectrum when the

DNA hybridized. A typical spectrum is shown in fig. 3.2.

Fig. 3.3 shows how the absorption at 260nm for DNA in 3mM phosphate buffer changes

with time. The time spans for hybridization ranged from 30min (10mM Tris buffer

with 50mM NaCl) to several hours (3mM phosphate buffer

). All hybridizations were

conducted at room temperature.

=2-amino-2-hydroxymethyl-1,3-propanediol, trivial name: trishydroxymethylaminomethane, molecu-

lar formula: C

1NO

. At 20

C the pK

value is 8.3, the pH is titrated to its final value by addition of

HCl

Phosphate buffer was prepared with monosodium phosphate, monohydrate (NaH

·H

O) and

disodium phosphate, heptahydrate (NaHPO

·7H

3.2. REQUIREMENTS FOR METALLIZATION

Figure 3.3: Hybridization of 48bp DNA in 3mM phosphate buffer. The change in absorption at

260nm with time indicates the transition from single to double stranded DNA.

3.2

Requirements for Metallization

Besides the presence of counter ions for the hybridization of the DNA, another requirement

for synthesis of metallized DNA structures is the appropriate choice of the pH value around

7: On the one hand DNA only hybridizes at moderate pH values, on the other hand the

reduction rate for silver ions strongly depends on the environment and increases in an

alkaline environment.

Repeating metallization experiments in various solutions for several times, indicates the

necessity of a buffer solution: Only in buffer solutions the peak positions, intensities and

widths are reproducible. This might also be related to the pH-dependence of the silver re-

duction, because in non-buffered solutions the precipitation of AgOH leads to fluctuations

of the pH value.

Furthermore, one has to avoid precipitates of insoluble silver compounds: First, these

precipitates diminish the concentration of silver ions available for the metallization and

therefore prevents reproducibility concerning the silver amount. Second, these precipitates

decrease the transmission of light due to scattering; for silver concentrations larger than

about 1mM this decrease is so pronounced, that recording a transmission spectrum has

become impossible.

For the photochemical method I tested different solutions, among them 10mM Tris buffer

with 50mM sodium chloride (pH 7.3), 3mM phosphate buffer, aqueous sodium nitrate

solution at pH 6 and aqueous sodium nitrate solution with sodium hydroxide at pH 8.3.

Tris buffer provided best results regarding reproducibility and the peak shapes.

For the so called Tollens reaction method, that is explained in 3.4, ammonium and sodium

hydroxide being present in the solution guarantee a basic environment. As the silver ions

are bound in a silver diamine complex, no precipitation occurs. Thus, an aqueous solution

CHAPTER 3. SYNTHESIS OF METALLIZED DNA

containing 5mM sodium nitrate for hybridization is sufficient.

3.3

Photochemical Metallization

Photochemical Metallization is a fast method for coating commercial DNA with silver.

This technique, which was first introduced by Berti for -phage-DNA [BAF05] exploits

the fact that UV light of 253nm oxidizes DNA bases.

Silver ions form complex bonds with DNA bases, when silver nitrate is added to a DNA so-

lution. When irradiating the sample with appropriate UV light, a redox reaction proceeds

in which the excess silver ions in solution are reduced to elementary silver. The silver-base

complexes act as nucleation seeds for this reduction and cluster growth along the DNA

starts. This process is schematically shown in fig. 3.4. According to [PWLW02] the bonds

between metals and DNA after irradiation are covalent. The photochemical DNA met-

allization is a fast and simple method. Its major drawback is, that no site-specific DNA

metallization is possible.

Figure 3.4: Principle of UV Metallization of DNA

For the experiments DNA with a length from 23 up to 96 basepairs, corresponding to

a length between 8nm and 33nm, and different sequences was provided by IBA GmbH,

Germany. The DNA was diluted to concentrations of 1µM in buffer solution (total volume

100µl) and hybridized at room temperature for several hours, depending on the buffer

solution. An amount of typically 100µM to 1mM silver nitrate, provided by Sigma-Aldrich,

Germany, was added. For comparison, the silver cluster concentration in the commercial

20nm silver sphere solution, mentioned in chapter 2.2.1 is 1.2nM. This corresponds to

300µmol silver atoms per liter. After several minutes the samples were put in front of an

UV hand lamp (Benda, 253nm, ca. 6W), the distance between the cuvettes and the lamp

was about 1cm. Typical irradiation time was 45min.

3.4

Tollens Metallization of Aldehyde-Modified DNA

The Tollens reaction is a redox reaction that is sensitive to reducing functional groups

such as aldehydes. By modifying DNA bases with aldehydes, this method allows for a site

3.4. TOLLENS METALLIZATION OF ALDEHYDE-MODIFIED DNA

selective metallization of the DNA as schematically shown in fig. 3.5.

Figure 3.5: Schematic of site selectively metallized DNA: Metallic nanoclusters are expected to

grow at certain aldehyde-modified sites only.

Glenn Burley from Thomas Carell's group at Ludwig-Maximilians-University, Munich,

provided different oligonucleotides with a length from 23 up to 38 basepairs, whereby a

fraction of thymine bases in the DNA sequence was modified with aldehyde groups.

The modified DNA can be either synthesized by solid-phase synthesis or by a polymerase

chain reaction (PCR). In a certain reaction, described in detail in a paper of G. Burley

[BGM

06], aldehyde groups are added to the T bases. Via an enzymatic incorporation

of modified nucleosides, acetylene reporter groups (see fig. 3.6) are inserted into certain

Figure 3.6: Acetylene molecule

positions of the oligonucleotide strands. These acetylene reporter groups react with alde-

hyde azides (see figure 3.7 by an Huisgen

1,3-cycloaddition click reaction[HBSG64]. This

Figure 3.7: Aldehyde azide

reaction is catalyzed by Cu(I). The product of this reduction is an oligonucleotide with

oxidizable aldehyde groups added to some pyrimidine bases as schematically shown in fig.

3.8.

Figure 3.8: Schematic of the modified DNA: Aldehyde groups are clicked to several bases of one

strand of the DNA

After the modified DNA had been hybridized in an aqueous solution containing a suffi-

cient concentration of sodium ions, Tollens reagent was added. Tollens reagent contains

silver nitrate and sodium hydroxide. A brownish precipitate, Ag

O, immediately falls

out. Adding ammonium dissolves this precipitate again and binds the silver ions in so

called silver diamine complexes, [Ag(NH

)

]

. Aldehyde groups as well as other reducing

sugar groups can reduce the silver ions in this complex to elementary silver. Thereby the

aldehyde groups are oxidized to a carboxylic acid as shown in fig. 3.9.

Two unsaturated systems can react by building up a ring structure. This class of reaction was mainly

investigated by Rolf Huisgen

CHAPTER 3. SYNTHESIS OF METALLIZED DNA

Figure 3.9: Chemical equation of the Tollens reaction: Silver ions are reduced to elementary

silver whereas aldehyde groups are oxidized

The alkaline environment is necessary so that the carboxylic acids are present in a depro-

tonated form; this shifts the equilibrium position to the side of the products [Blu03].

As can be seen in the formula each aldehyde group can reduce two silver ions. The excess

silver ions can only be reduced in presence of a reducing agent, such as a development

solution containing formaldehyde. This development process will not produce new silver

clusters, but enlarges the already available seeds.

3.5

Materials Section

All chemicals were provided by Sigma-Aldrich. DI Water was provided by Millipore. The

DNA sequences are listed in the Appendix A. All hybridization and metallization was

done in disposable Brand UV cuvettes at room temperature.

Particular attention should be paid to the used cuvettes: When conducting the classical

silver mirror reaction, where Tollens reagent is added to a glucose solution, the elementary

silver sticks to the borders of a glass vial as it acts as an ion exchanger and attracts the

silver ions. In the experiments of this work, however, it is assumed that the positively

charged silver complexes are attracted by the negatively charged DNA molecules and not

by the glass. This is confirmed by Braun, who mentions that DNA can work as an ion

exchanger [BESBY98].

To achieve certainty that the reaction is carried out on DNA instead of the cuvette

window, a metallization process was done in a Hellma QS cuvette as well as a Brand

plastic UV cuvette: The spectra for the two samples were almost identical indicating that

the cuvette does not influence the reaction. Afterward the solution with the metallized

DNA was removed from the glass cuvette and it was dried with nitrogen gas: Now the

UV/vis spectrum showed no plasmon peak any more. This is a strong indication that the

silver precipitates on the DNA in solution and not on the glass surface.

Nevertheless, to avoid any competing processes plastic cuvettes were used instead of glass

ware.

Chapter 4

Absorption Spectrometry

In this chapter the principle of absorption spectrometry will be shown. Plasmon peaks

of metallized DNA will be investigated for UV- as well as Tollens-metallized samples.

Therefor several synthesis parameters were systematically varied. The experimental results

are compared to theoretical calculation.

4.1

Principle of Absorption Spectrometry

Absorption spectrometry, also known as UV/vis spectrometry, is a widely used tool for

metallic nanoparticle characterization as it shows the plasmon absorption peaks.

The working principle of this method is: A light source consisting of a tungsten halogen

lamp and a deuterium lamp irradiates the cuvette. A grating in the beam path adjusts

the wavelength and provides for monochromacity, the wavelength of the irradiating beam

can thereby be tuned over a wide range, from the UV to the near infrared spectral region.

Inside the solution the light intensity is attenuated according to the Lambert-Beer relation

trans

= I

-D

with the path length D, the extinction coefficient = N

ext

. N is the

density,

ext

is the extinction cross section, introduced in 2.2.1. A detector measures the

transmitted intensity. A schematic is shown in fig. 4.1.

By using the relation 'T+E=1', whereby 'T' is the transmission and 'E' is the extinction,

Figure 4.1: Principle of UV/vis spectrometry

CHAPTER 4. ABSORPTION SPECTROMETRY

the latter can be calculated. Extinction is the sum of absorption and scattering. As the

metallized DNA molecules are only a few nanometers in size, scattering is negligible for

the low light intensities in the spectrometer due to the Rayleigh approximation, described

in chapter 2.2.1. Thus in the following the term 'absorption spectrum' will be mostly used,

although some contribution to the extinction might come from scattering, i.e. by larger

particles available in the solutions. This notation points up that absorption of light is the

predominant effect we are looking for when applying this technique.

For the experiments a Lambda 900 two-beam spectrometer of Perkin Elmer was used.

Contrary to the simplified description above, now two beams are employed: The first beam

transverses the cuvette filled with the solution. The second beam, that goes just through

air and an aperture, compensates for any fluctuation. All spectra were normalized by the

absorption of a cuvette filled with water/buffer-solution. The reason for this normalization

is, that the plastic cuvettes partly absorb in the UV and therefore overlap the DNA

absorption.

4.2

Absorption Measurements of UV-Metallized Sam-

ples

Different short oligonucleotides were metallized using the UV method as described in

chapter 3.3. Thereby various parameters such as lengths and sequences of the oligonu-

cleotides, the irradiation time and the silver concentration were systematically varied.

Afterward the samples were characterized by UV/vis spectrometry.

Before metallization, the UV/vis spectra look like the following: The DNA in solution

exhibits a peak around 260nm due to the absorption of the bases. When adding silver

nitrate, the peak intensity increases and shifts to the red

. When ions, that form insoluble

silver compounds, such as chloride, phosphate or hydroxide, are present in the solution,

the extinction increases due to scattering of light by the precipitate. As discussed in 2.2.1

this extinction is proportional to

-4

. These spectra before metallization are shown in fig.

4.2.

Metallization of the DNA, i.e. the growth of nanoclusters on the DNA-template can be

monitored by the appearance of a plasmon peak between 400 and 450nm, such as shown

for commercial silver spheres in fig. 2.4.

Unless otherwise noted, the following measurements are conducted in 10mM Tris buffer

with 50mM NaCl at pH 7.3. Hybridization at room temperature was completed after 90

minutes at the latest. The oligonucleotides are always brought to final concentrations of

1µM. Unless otherwise noted the amount of added silver was 100µM, corresponding to a

silver-to-base of 1:1. Typical irradiation times range from 30 to 45 minutes.

The reason is that nitrate absorbs light around 300nm. The absorption peaks of the DNA and the

nitrate cannot be resolved in the spectra.

4.2. ABSORPTION MEASUREMENTS OF UV-METALLIZED

SAMPLES

Figure 4.2: Absorption spectrum of a Tris buffer solution with 1µM 48bp dsDNA before and

after addition of silver. Details about the different contributions to the absorption can be found

in the text.

4.2.1

Influence of pH

To find out optimum synthesis conditions, metallization measurements were done in so-

lutions with varying pH values.

Fig. 4.3 shows the UV/vis spectra of UV metallized DNA in pure water titrated to certain

pH values by adding NaOH

As the pH measurements were done with litmus paper, the values are just rough estima-

tions. The DNA is present as single strands, because the aqueous solution contains too

few counter ions. The ratio of silver ions to DNA bases is 1:1, the irradiation time in this

measurement is approximately 30 minutes.

Only in an alkaline environment, a plasmon peak appears. The peak intensity increases

with the pH value, the peak position shifts to the blue suggesting a smaller average particle

size. There is also a significant rise in the background extinction when increasing the pH.

This is caused by precipitated silverhydroxide, AgOH.

The reduction potential of silver is pH-dependent, therefore the reaction rate is also pH-

dependent. This was experimentally shown here: A pH above 7 is necessary to provide a

plasmon peak. As silver ions can bind OH

ions by forming silver hydroxide and therefore

lower the pH-value, a buffer solution is recommended for the UV-metallization. These

results support the use of Tris buffer at 7.3 as a solution for the UV-metallization synthesis.

The pH value of pure water was only 6. This probably originates from carbonic acids in water that

occur when carbon dioxide from air is taken up in solution.

CHAPTER 4. ABSORPTION SPECTROMETRY

Figure 4.3: Influence of the plasmon peaks of UV-metallized DNA on the pH value in solution.

Only for alkaline environments plasmon peaks show up.

4.2.2

DNA Sequence

Hybridization

As the persistence length of single-stranded DNA (ssDNA) is much shorter than that of

double-stranded DNA (dsDNA), different cluster shapes, especially different asphericities

of the nanoparticles, are expected. Therefore UV metallization experiments were carried

out on ss as well as dsDNA.

Silver was added to 48mer DNA in an Ag-to-base ratio of 1:1. The samples were irradiated

for 30min. The spectra can be seen in fig. 4.4, the results are summed up in the following

table:

Table 4.1: Absorption measurements of UV-metallized ss and dsDNA

ssDNA

dsDNA

plasm

404nm

409nm

plasm

25.9%

33.4 %

FWHM

80nm

86nm

background

16%

18 %

DN A

262nm

262 nm

DN A

77.3%

68.1%

plasm

is thereby the position of the plasmon peak. Its intensity is denoted by I

plasm

4.2. ABSORPTION MEASUREMENTS OF UV-METALLIZED

SAMPLES

Figure 4.4: Comparison of UV-metallization for single- and double-stranded DNA

given relative to the intensity of the incoming beam. FWHM, the 'Full Width at Half

Maximum' is a characterization for the broadness, it is given in nm. I

background

is the

background absorption, read off as absorption at long wavelengths, i.e. 700nm. It is given

relative to the incoming intensity.

DN A

gives the peak position for the absorption of the

bases, its intensity I

DN A

is again relative to the incoming beam. These denotations will

be also used for the following analysis.

The intensity of the plasmon peak in the spectrum is 1.5 times higher in the case of the

dsDNA. The concentration of DNA molecules in the single-stranded solution is, however,

twice as large as that in the double-stranded solution

The single-stranded sample might

therefor contain more clusters, whereas the clusters in the double-stranded solution might

be larger, assuming that all DNA molecules are involved in the metallization process and

all silver atoms become reduced. Thus the intensity is not applicable for comparing the two

systems. The plasmon peak for the dsDNA is slightly red-shifted compared to the ssDNA,

indicating larger particles. This originates either from the lower DNA concentration as

discussed above or from the larger dimensions of dsDNA due to its long persistence length.

The absorption of the DNA bases at 262nm is higher for the dsDNA than for the ssDNA.

The reason of this is the composition of the bases: The special sequence of the used

The reason is simply that the amount of bases was kept constant in solution. The double-stranded

DNA molecules contain twice as much bases as the single-stranded DNA molecules.

This is not a contradiction to Tinoco's theory presented in 3.1 as the DNA sequences in the two

solutions are not identical: The ssDNA consists of 2µM of the 48mer DNA sequence listed in B, whereas

the dsDNA consists of 1µM of this sequence and 1µM of the complementary sequence. In general, DNA

sequences do not have the same absorption coefficients as their complementary sequences.

CHAPTER 4. ABSORPTION SPECTROMETRY

48 basepair (=48mer, 48bp) absorbs less than its complementary strand. There is no

difference in the background extinction. The peak width is slightly larger for double-

stranded DNA.

Thus, there are slight differences between metallized ss and dsDNA. The red-shift of the

plasmon peak indicates that metallized dsDNA is larger than metallized ssDNA. This

accords to the expectations.

Base Sequence

To investigate the influence of the base sequence on the outcome of the metallization

process, three different 48mer dsDNA samples were metallized with fractions of G- and

C-bases of 0%, 48.5% and 75%. Fig. 4.5 shows the specra, the table lists the data:

Figure 4.5: UV/vis spectra for different 48mer oligonucletide sequences. All bases can be metal-

lized. The exact peak position as well as the intensity depend on the specific sequence, however.

Table 4.2: Absorption measurements of UV-metallized DNA with different GC-fractions

GC content

48.5 %

75%

plasm

415nm

410nm

425nm

plasm

54%

34%

55%

FWHM

99nm

87nm

126nm

background

26%

17%

22%

DN A

263.5nm

262nm

259.6nm

DN A

86%

77%

83%

In contrast to low and medium GC-contents, the 75%GC-peak is quite asymmetric, in-

4.2. ABSORPTION MEASUREMENTS OF UV-METALLIZED

SAMPLES

dicating a very broad particle size dispersion according to Henglein [HG99]. The peak

intensity of the 48.5% GC-sample is significantly lower than for the other samples. This

is a reproducible effect that must be attributed to the special sequence of this sample.

The background intensities are approximately proportional to the peak intensities. The

positions and intensities for the DNA-nitrate-peak cannot be compared in this case, as

different bases show different absorption spectra. The peak position seems to depend on

the base sequence, but there is no general trend observable.

The measurements showed, that all bases can in general be metallized by the UV-method.

The peak intensities, positions and shapes, however, depend crucially on the DNA se-

quence. These observed differences indicate different particle sizes for the different base

sequences. As there is no general relation between the peak position or bandwidth and

the GC-content, the metallization probably does not only depend on the bases themselves

but also on the surrounding sequence.

4.2.3

Silver Amount

To observe the size and shape dependence of the particles on the silver concentration, the

amount of silver ions was varied between 10µM and 0.5mM, corresponding to silver-to-

base ratios between 0.1 and 5. The samples with 48mer DNA and silver were irradiated

for 45min.

Figure 4.6: UV/vis spectra of UV metallized DNA with various Ag concentrations

CHAPTER 4. ABSORPTION SPECTROMETRY

Figure 4.7: Blue shift of the plasmon peak when increasing the silver amount

Table 4.3: Absorption measurements of UV-metallized DNA for various silver concentrations

ratio Ag/bp

0.1

0.5

plasm

416nm

413nm

408nm

405nm

plasm

6.4%

22.6%

43.3%

90.8%

FWHM

94nm

96nm

110nm

background

17%

30%

DN A

262nm

DN A

77.4%

79.4%

80.8%

86.4%

The spectra show a distinguishable peak around 410nm for silver-to-base-ratios larger

than roughly 0.5. For silver-to-base ratios larger than about 10, a significant precipitation

of silver chloride reduces the transmittance of the sample and thereby makes the extinction

spectrum less reproducible. Thus only low silver-to-base-ratios shall be analyzed here.

The position of the nanoparticle peak shifts to the blue when increasing the silver-to-base

ratio. That is also shown in fig. 4.7. This trend still holds good after deconvoluting the

spectrum to eliminate to influence of AgCl scattering which is proportional to

-45

. The

interpretation of this blue-shift is given in 4.2.7.

One would expect the DNA peaks to shift to the red due to an overlap with the increasing

nitrate peak. This cannot be detected. A possible explanation is that more silver entails

more complexation. Silver-DNA-complexes, however, cause a blue shift of the base absorp-

tion peak [ES68]. Thus, the two effects might compensate for each other. The increasing

intensity of the DNA peak, that can be detected for large silver concentrations, traces

back to the increasing amount of nitrate ions in the solution.

The exact deconvolution method will be explained in 4.2.7. Deconvolution is necessary for the peak

determination as the plasmon spectrum is overlapped by the AgCl scattering spectrum, that depends on

the amount of added silver.

4.2. ABSORPTION MEASUREMENTS OF UV-METALLIZED

SAMPLES

With increasing silver amount, the intensity of the nanoparticle peak increases from 6.7%

at ratio 0.1 to 90.8% for ratio 5. This indicates either a greater number of metallized

particles or larger clusters. There is also an increase in the extinction background from

3% to 30%. This might originate from unspecific silver deposition in the sample, that

increases with the silver amount. For silver-to-base ratios not larger than 1 the FWHM

is roughly 70nm. When increasing the silver concentration by a factor of five, the peak

width grows 37% to a value of 96%, indicating a huge change in the particle size or a

bigger polydispersity of the particle sizes.

Summarizing it can be stated that plasmon peaks occur already for very small silver-to-

base-ratios. The peak intensity increases with the silver amount, a saturation value was

not observed. Thus more silver ions means either more or larger particles. The discussion

about the tendency of the peak position to shift to the blue with increasing silver-to-base

ratios will be discussed later, in 4.2.7.

4.2.4

Irradiation Time

To gain information about the reaction kinetics, the irradiation time for a 48mer sam-

ple with a silver-to-base ratio 1:1 was varied from 5min to 150min. Fig. 4.8 shows the

corresponding spectra.

Table 4.4: Absorption measurements of UV-metallized DNA as a function of the irradiation time

irradiation time

5min

15min

30min

150min

plasm

420nm

414 nm

410nm

404nm

plasm

55.5%

52.5%

49.5%

31.7%

FWHM

126nm

96nm

94nm

86nm

background

25%

22%

DN A

260nm

261.5nm

216.7nm

262nm

DN A

84%

80.7%

80%

71%

For all irradiation times a plasmon peak between 400 and 420nm is observable in the

UV/vis spectrum. With increasing time, the peak position shifts from 420nm to 404 nm

as shown in fig. 4.9. The discussion about this will be delayed to 4.2.7. In 4.2.6 it is shown

that the UV-Metallization does not induce breakage of DNA strands, thus this cannot be

the origin for the blue shift.

The peak intensity decreases with time from 55.3% to 31.5%. The background i.e. the

constant absorption at long wavelengths, remains relatively constant during the measure-

ment. The broadness of the peak (FWHM) decreases significantly from 126nm at 5min

to 86nm at 150min.

The intensity of the DNA-nitrate-peak decreases with time. The main reason for this is

that the bases get more and more covered by silver which partially screens them from

absorbing UV-light. A second contribution is a reduced overlap with the plasmon peak

CHAPTER 4. ABSORPTION SPECTROMETRY

Figure 4.8: UV/vis spectra of UV metallized DNA with various irradiation times

Figure 4.9: Blue shift of the plasmon peak when increasing the irradiation time at UV metal-

lization

4.2. ABSORPTION MEASUREMENTS OF UV-METALLIZED

SAMPLES

due to narrower peaks.

The metallization reaction seems to set in immediately with UV-irradiation as a pro-

nounced plasmon peak already occurs after short irradiating times. This is also confirmed

by the observation that a change in color from achromatic to light yellow instantaneously

sets in when irradiating the sample. In the first 60 to 90hours (due to clarity not all

measured graphs are shown) the peak positions, bandwidths and intensities change with

time. Spectra taken after 90min and 150min are similar indicating that the reaction has

been completed after approximately one hour.

4.2.5

DNA Length

As the absorption spectrum of a metallic nanorod depends on its length, the influence of

the DNA length on the plasmon peaks shall be investigated. Therefor the DNA length

was varied between 23bp and 96bp, corresponding to 7.8nm and 3.2.6nm.

Figure 4.10: UV/vis spectra for UV-metallized oligonucleotides of varying length

CHAPTER 4. ABSORPTION SPECTROMETRY

Table 4.5: Absorption measurements of metallized DNA with various lengths

length

23bp

38bp

48bp

96bp

GC-content

65.2%

57.9%

48.5%

47.9%

plasm

413nm

408nm

421nm

plasm

47.2

33.4

49.7

FWHM

101

124

background

17%

17 %

17%

21%

DN A

261nm

262nm

261nm

DN A

68.2%

70.8%

77%

94.6%

There are slight, reproducible differences between the individual spectra, listed in the

table above. The spectrum of the 23mer oligonucleotides is almost identical to that of the

38mer. This can be easily understood regarding the sequences as listed in Appendix A:

They both have the same starting and ending sequence. The intermediate part consisting

of 15 basepairs occurs once in the 23mer whereas it is repeated in the 38mer. The 48mer

spectrum does not fit to the other sequences as it is (reproducibly) much lower in intensity

and slightly red-shifted. This might trace back to the specific sequence of the 48mer as the

same observations were made when comparing the 48mer sequence to 48mer sequences

with different GC-contents in chapter 4.2. For the other sequences, there is a slight red-

shift of the peak with the DNA length, indicating larger particles. The fact, that a very

small quadrupole shoulder at 350nm can be discerned in the 96mer spectrum confirms

this conclusion.

In summary, samples with different length show slight differences in the absorption spec-

tra. A possible influence of the DNA length on the spectrum is, however, overlapped

by the sequence-dependence of the UV-metallization. Thus DNA molecules of various

lengths with similar DNA sequences have to be produced and analyzed to draw further

conclusions about the influence of the DNA length.

4.2.6

Reference Samples

Irradiation of samples with 10mM Tris buffer solution and silver nitrate shows a charac-

teristic structure: Below 320nm the transmission is lowered due to the absorption of the

nitrate present in the solution. Above 320nm the transmission is lowered due to unspecific

precipitation of silver chloride that occasionally occurs. A UV/vis spectrum of this can be

seen in the appendix B. This interpretation is confirmed by the way, that the spectrum

changes when varying the amount of added silver.

When a solution with DNA but without silver is irradiated, the spectrum does not change.

This is also an indication that the UV lamp does not break DNA strands - otherwise the

absorption would change according to 3.1.

The UV-irradiation is implicitly necessary for the particle synthesis, without it no plas-

mon peak occurs, not even after some hours. According to manufacturer information the

4.2. ABSORPTION MEASUREMENTS OF UV-METALLIZED

SAMPLES

Figure 4.11: Theoretical calculation of silver plasmon peaks in water, done by Slistan-Grijalva

et al. [SGHURS

05] Small particles mainly exhibit an enhancement of the absorbance as well

as a narrowing of the peak with increasing size. Larger particles show a red shift with increasing

size because of retardation effects.

intensity of the UV/vis spectrometer is not sufficient to reduce silver ions and induce

nanoparticle growth.

Thus, plasmon peaks only occur if silver as well as DNA is present in solution and the

sample is irradiated.

4.2.7

Discussion

Comparison with Theory

The experimental values shall now be compared to theoretical calculations done by Slistan-

Grijalva [SGHURS

05] et al.. They applied Mie theory up to the tenth multipole for

silver nanospheres in water. Although the metallized DNA structures are not assumed to

be spherical particles, the experimental and theoretical values shall be compared to get a

first insight about the processes happening during metallization. The theoretical values,

that are shown in fig. 4.11 and 4.12, agree with experiments I did with commercial silver

spheres in a range from 20 to 80nm in diameter, shown in fig. 4.13.

For small particles, the peak position remains approximately constant. Small red shifts

with increasing size are due to a radius-dependent dielectric function. Very small parti-

cles show broad peaks: In bulk medium the mean free path of electrons is 52nm. Thus

for particles with a radius shorter than 26nm an additional damping mechanism, surface

scattering, becomes important and increases the peak width. According to the electro-

static calculation in 2.2.1, the peak intensity is proportional to the volume. This is at least

quantitatively in accordance with Slistan-Grijalva's results for small particles. Theory pre-

dicts a significant red shift in the plasmon spectrum for larger particles since retardation

effects, such as screening of the incoming wave, shift the plasmon peak to the red. The

Experimental results for the maximum absorbance in dependence on the particle size are not shown as

the concentrations of the commercial particles are size-dependent. Thus the intensities are not comparable.

CHAPTER 4. ABSORPTION SPECTROMETRY

(a) maximum absorbance

(b) bandwidth

Figure 4.12: According to Slistan-Grijalva's calculations [SGHURS

05], particles in water with

a radius of 20nm show a maximum plasmon absorbance twice as large as 3nm particles. The

bandwidth remains almost constant for particles with sizes from 10 to 25nm. For small and large

particles, several damping mechanisms broaden the peak. Only the graphs for water, not the

graphs for ethylene glycol are relevant for the comparison!

(a) plasmon peak position

(b) FWHM

Figure 4.13: Experimental values for the plasmon peak position and the bandwidth in depen-

dence on the particle size. extracted from 2.4 Measurements were done with commercial silver

spheres described in the text.

4.2. ABSORPTION MEASUREMENTS OF UV-METALLIZED

SAMPLES

excitation of higher order modes, such as the quadrupole mode, broadens the spectrum.

One aspect Slistan-Grijalva does not discuss, is polydispersity of the particle sizes. Thus

experimental spectra will certainly show broader spectra than Slistan-Grijalva predicts.

Another difference between my measurement conditions and Slistan-Grijalvas calculations

is the refractive index of the surrounding medium. Slistan-Grijalva chose water and there-

fore a refractive index of 1.33. In the UV-metallization experiments, the aqueous solution

contains various amounts of salt; the refractive index is therefore slightly increased. More-

over the influence of the DNA, namely its refractive index and the core-shell character

of the resulting nanoparticles must not be ignored. The refractive index influences the

absolute values of the peak position, the maximum absorbance as well as the bandwidth.

The result of the UV metallization is, that the metallization process worked for all DNA

samples. All spectra showed plasmon peaks in the same range and with similar shapes

and widths. There were slight, reproducible differences, indeed, that shall be concluded

and discussed in the following:

All measurements show plasmon peaks in the range between 405 and 425nm. As the re-

fractive indexes of neither the buffer solution nor the DNA are known, the values cannot

be quantitatively compared to the theoretical values. It can be only concluded that the

particle radius is probably below 20nm. Taking the peak position as the sole indicator for

particle size, the smallest particles, corresponding to the shortest peak wavelengths, oc-

curs for ssDNA, for high silver concentrations and long irradiation times. The former can

be understood as ssDNA has a shorter persistence length than dsDNA and therefor forms

bundles that are at least in one direction smaller than the dsDNA. The latter cannot be

understood with this simple theory and will be explained below. A possible explanation

for the third one is that the DNA strands break during irradiation and therefore the par-

ticle size decreases. An alternative explanation will be given below. The largest particle

sizes occur for long DNA, short irradiation times and very high GC-contents. The large

particle size for long DNA matches the expectations as long DNA means more seed posi-

tions and therefore larger nanoparticles. The large size for short irradiation times cannot

be explained with this theory, only conjectures can be made about its origin. The large

particle size for high GC-contents shows that the metallization is, indeed, sequence depen-

dent. The FWHM is mostly between 80 and 100nm. Assuming complete monodispersity,

which certainly does not hold in reality, this means diameters below 9 or above 70nm

according to Slistan-Grijalva. The smaller size is more in accordance with the results of

the peak positions and therefore more probable. In reality, however, polydispersity, the

deviations of the refractive index from that of water as well as shape effects may influ-

ence the FWHM. Most peaks were quite symmetric, exceptions are the 75%GC-sample,

samples that were irradiated for a very short time and samples with long DNA. Accord-

ing to Henglein [HG99] an asymmetric peak shape indicates a broad size distribution of

particles. The reverse, however, is not always true. A slight shoulder is detectable for the

75% GC sample which might be indicative of a quadrupole mode as predicted for large

(60nm)particles. On the other hand it might reflect the particle asymmetry; a definite

assignment is not possible at this stage. The background extinction, that can be assigned

CHAPTER 4. ABSORPTION SPECTROMETRY

to extinction by unspecific silver deposition is between 2 and 30% and depends e.g. on

the amount of added silver ions. There might be a tendency for a red-shift and a broad-

ening of the plasmon peaks with the length of the DNA. To prove this, it is necessary to

build up similar DNA sequences of various lengths, as the sequence strongly influences the

metallization process. When increasing the amount of silver the absorption intensity as

well as the bandwidth increases, when increasing the irradiation time, the absorption in-

tensity as well as the bandwidth increases. Regarding the calculations of Slistan-Grijalva,

nor of them can be explained by simple Mie theory as the absorption intensity and the

bandwidth always show opposite trends. Hence a more advanced theory concerning about

particle shapes, as will be discussed later, has to be applied.

As a conclusion, the comparison of the measured values and the Mie theory calculated

by Slistan-Grijalva show that small nanoparticles have been produces. The peak position

depends on the length and sequence of the DNA as well as the measurement conditions.

The results cannot be completely explained in a quantitative way and thus another theory

has to be applied.

Rodlike Character

As we assume the metallized DNA to have either rod-like (whole DNA is metallized)

or chain-like (nanoclusters have grown on DNA) character, two extinction peaks should

appear in the spectrum. The electrostatic calculation for nanorods from 2.3.1 predicts a

low intensity peak at short wavelengths around 400nm as well as a high intensity peak

at long wavelengths. The latter one can be anywhere in the visible or near infrared range

and strongly depends on the axis ratio. A similar behavior is also expected for chains of

nanoparticles [RABdA06], as they might have grown on the DNA strands. Experiments

with nanorods in solution, as for example done by C. Soennichsen's group [BKRS06] are

in great accordance with this theory.

Recorded absorption spectra of the metallized DNA, however, have not shown a dominant

long-wavelength peak, even when the scan range was extended up to 1200nm.

Literature research, however, shows that not all plasmon peaks obtained for nanorods are

in the wavelength range specified by the electrostatic calculation as mentioned above:

E.g. in 1989, Goudonnet [GBP89] analyzed silver spheroids with a length of 12nm and a

diameter of 3.6nm on a quartz substrate. He identified two peaks in the spectrum, one at

468nm and one at 338nm. Qu [QD05] found a 350nm plasmon peak for silver nanoparticles

grown on a copper foil by silver mirror reaction. In his PhD thesis, M. Noyong [Noy05]

calculated the plasmon spectrum for different aggregates of nine silver spheres with a

diameter of 40nm. He found one peak around 350nm and another one between 450nm

and 500nm.

Possible low-intensity absorption peaks of metallized DNA in a range below 400nm might

be covered by the absorption of the DNA and the nitrate. Thus the spectra were decon-

voluted

to eliminate all peaks that are not related to plasmon oscillations. The result of

The transmission spectrum after the irradiation was divided by the spectrum before irradiation.

Therefore the influence of DNA, salts and silver chloride precipitation is eliminated. Afterward the spectra

4.2. ABSORPTION MEASUREMENTS OF UV-METALLIZED

SAMPLES

Figure 4.14: Relative absorption of UV-metallized DNA of various lengths. The spectrum is

a deconvolution of the measured data with the data just before nanoparticle growth. As this

spectrum shows just the relative absorption, also negative values are possible: As explained

already above, the absorption of the DNA bases decreases during the metallization process,

therefore the relative absorption is below zero.

such a deconvolution can be seen in fig. 4.14. Indeed, there are two peaks visible: One

at 300nm and one at 420nm, which can be assigned to the transverse and longitudinal

plasmon respectively.

In the spectra the long wavelength peak is more intense and more sensitive to changes in

the synthesis parameters than the short wavelength peak. This is in accordance with the

electrostatic calculation. The following table lists the peak positions found for deconvo-

luted data.

was plotted as an absorption spectrum. The correction was done with the transmission spectra to avoid

division by zero

CHAPTER 4. ABSORPTION SPECTROMETRY

Table 4.6: Peak positions after deconvolution

length

23bp

313nm

416nm

38bp

313nm

420nm

48bp

314nm

415nm

96bp

307nm

423nm

sqeuence

0% GC

302nm

419nm

48.5% GC

313nm

415nm

75% GC

306nm

428nm

irr. time

5min

302nm

423nm

15min

306nm

415nm

30min

308nm

413nm

45min

312nm

411nm

90min

314nm

407nm

150min

313nm

407nm

Ag-base ratio

0.5:1

307nm

416nm

1:1

312nm

412nm

5:1

312nm

404nm

The longer the DNA, the more separated the peaks are, i.e. the more elongated the

particle is. This is again not true for the 48mer DNA. The 48mer sequences with different

GC-values also exhibit two peaks. There is, however, no trend observable. The longer

the samples are irradiated, the closer the peaks approach. This indicates a decrease of

the particles' axis ratio: The maximum length of the nanoparticles is determined by the

length of the DNA and therefore does not change during metallization. With increasing

irradiation time, more silver is deposited on the DNA, the particles become rounder. A

similar explanation can be used to explain the peak shifts dependent on the silver-to-base

amount: The more silver is added, the more spherical the particle becomes.

Thus, both peaks could be identified in the spectrum but contrary to the electrostatic

calculation, they are tremendously blue-shifted: One possible reason for the blue shift

of the long wavelength plasmon is the charge of the nanoparticles. According to Mul-

vaney [MPJG

06] the injection of electrons to the surface shifts the longitudinal plasmon

50nm/V to the red. This effect is more pronounced for rods than for spheres, as the en-

hanced oscillator strength makes the particle be more prone to surface perturbations. It

also leads to a damping of the peak.

Concerning the literature mentioned above, the blue shifts of both plasmon peaks mainly

occurs for particles in contact with a surface. The 'surface' in this experiment is the DNA.

Unfortunately there is no theory in literature explaining this effect up to now.

Summing up it can be said, that small rod- or chainlike metallic nanoparticles have grown

4.3. ABSORPTION MEASUREMENTS OF TOLLENS-METALLIZED

SAMPLES

on DNA templates. Their axis ratio can be tuned by varying the length, by the added

amount of silver and by the irradiation time. Unfortunately no quantitative theory is

applicable to extract the axis ratios from the optical data.

4.3

Absorption Measurements of Tollens-Metallized

Samples

The following section covers the optical properties of Tollens-metallized DNA. Aldehyde-

modified oligonucleotides are therefor metallized in solution by the Tollens method de-

scribed in 3.4 and investigated. The analysis method, UV/vis absorption spectrometry,

is the same as for UV-metallized DNA. Parameters such as the length, the modification

density and the silver amount are varied systematically. Moreover the temporal evolution

of the growth as well as the influence of a development solution are investigated.

In contrary to the UV metallization process, where a buffer is necessary to guarantee

for good reproducibility, the solution for the Tollens metallization must not buffer at all.

This is, because the Tollens reaction works best in alkaline environments and therefore the

Tollens reagent itself has a pH value above 10, depending on its dilution. DNA however

cannot be hybridized under extreme pH conditions.

. If DNA was hybridized in a buffered

solution with a pH significantly lower than 9, the environment would not be basic enough

for the reaction, even after the addition of the Tollens solution. Thus, to fulfill all these

requirements, the modified DNA was hybridized in an aqueous solution containing 10mM

NaNO

. The samples were kept at room temperature overnight to provide enough time

for complete hybridization. Afterward an aliquot of a Tollens reagent was added to the

solution and raised the pH to the required pH above 9.

For metallization, three different oligonucleotides were provided: A 38 basepair and two

different 23 basepair oligonucleotides. The DNA consisted of 30 basepair and 15 base-

pair long middle parts, respectively, of which every third or fifth base is modified. The

denotation for the oligonucleotides is first the length of the middle part and second the

density: 15-5 means a 23 basepair DNA containing a middle part where every fifth atom

is aldehyde modified. The available oligonucleotides therefore are denoted as '30-3', '15-

5', '15-3'. It is important to notice, that only one strand of the dsDNA is modified. A

schematic of the different oligonucleotides is shown in fig. 4.15.

Composition of Tollens Reagent

The Tollens reagent used for the experiments, unless otherwise noted, contains 317µM

AgNO

, 37.5µM NaOH and 200µl of a 28% ammonium hydroxide solution and was filled

up with water to reach a final volume of 1ml.

The outcome of the experiment depends crucially on the amount of the ammonium: Too

This was tested with 1µM 48mer DNA in an aqueous solution with 10mM NaNO

to which NaOH

was added until a pH value of 9.5 was reached: No hypochromism was observed indicating that the DNA

was not hybridized

CHAPTER 4. ABSORPTION SPECTROMETRY

Figure 4.15: Simple schematic of aldehyde modified dsDNA, for the the utilized samples 15-3,

15-5 and 30-3. Each black vertical line indicates a basepair, red disc indicates an aldehyde-

modification.

little ammonium cannot dissolve all the silver oxide precipitation. Too much ammonium,

however, drastically slows the reaction.

When using a Tollens reagent containing 450µl of the ammonium solution instead of

200µl, it lasts between 24 and 72 hours until a reaction takes place. The peak that occurs

is not intense, only 0.06% of the light is absorbed. The spectra show differences between

samples containing modified and that containing unmodified DNA, but these differences

are not very pronounced, indeed. This is shown in fig. 4.16. When decreasing the ammo-

nium amount to the 200µl mentioned already above, a significant plasmonic peak already

appears after several minutes for the modified DNA. After some hours the velocity of the

reaction slows down. Various spectra are shown in the next paragraphs.

Reference Samples

There is also a very small peak in the spectrum, when Tollens reagent is added to un-

modified DNA, that can be attributed to electrostatic attraction of the silver ions to the

negatively charged phosphate backbone. A 10mM NaNO

solution with an aliquot of the

Tollens reagent containing no DNA did not show any plasmonic peak at all. Reference

samples such as B.4 can be found in Appendix B.

4.3.1

Absorption Measurements of Tollens-Metallized dsDNA

Modified dsDNA shall now be Tollens-metallized and afterward be characterized with

respect to its optical properties.

As described above, modified oligonucleotides were hybridized in an aqueous NaNO

so-

lution and afterwards provided with aliquots of a Tollens solution poor in ammonium.

Figure 4.17 shows the temporal evolution of a plasmon peak after addition of Tollens.

The peak absorbance increases with time and finally approaches a maximum value (not

shown in the figure). The peak position shifts to the blue with time. There is no general

trend for the bandwidth: Whereas the 30-3 samples broaden with time from around 100nm

4.3. ABSORPTION MEASUREMENTS OF TOLLENS-METALLIZED

SAMPLES

Figure 4.16: UV/vis spectra 72 hours after addition of an ammonium-rich Tollens reagent to

three different samples. For modified 30-3 DNA a weak peak has developed. The sample with

unmodified DNA shows a slight increase in absorption at the same wavelength as can be seen in

the zoom. The absorption of the sample containing no DNA is also enhanced without showing

any peak structure.

CHAPTER 4. ABSORPTION SPECTROMETRY

Figure 4.17: Temporal evolution of the plasmon peak for Tollens metallization. The silver amount

(32mM) was chosen in the saturation regime, the dsDNA center parts lengths was 15 basepairs;

every third basepair was aldehyde-modified.

to 113nm, the 15-3 sample seems to decrease its HWHM

from 95nm to 91nm.

The increasing intensity indicates either more or larger particles. This matches the expec-

tations as the particles are monitored during their growth process. This is discussed in

more detail in 4.3.4. The blue shift with time reminds of the blue shift of UV-metallized

DNA with increasing irradiation time. Contrary to the UV metallization, a deconvolu-

tion of the spectrum some time after the Tollens addition with the spectra directly after

the Tollens addition does not reveal a second peak. For wavelengths shorter than 350nm

absorption can be attributed to the Tollens reagent. The inexplicable fluctuation in the

FWHM may be attributed to inaccuracies in evaluating the data.

After keeping the samples overnight at room temperature, the mentioned trends are not

valid any more: The peak shifts to the red and gains disproportionately high intensity.

This tendency suggests the aggregation of the nanoparticles in solution. A further growth

of the clusters is not probable, as a saturation was already observed some hours after

preparation.

Contrary to the UV-metallized samples now the Half-Width-Half-Maximum (HWHM) is given: Due

to an overlap with the DNA/nitrate peak the FWHM cannot be read off for the broad peaks of Tollens-

metallized samples. As one cannot assume a symmetric particle shape either, the HWHM is given instead

of the FWHM.

4.3. ABSORPTION MEASUREMENTS OF TOLLENS-METALLIZED

SAMPLES

Fig. 4.18 shows the UV/vis spectra for different modified oligonucleotides five hours after

addition of two different Tollens concentrations.

Figure 4.18: The three oligonucleotide types described in the text 5 hours after addition of two

different amounts of Tollens solution

The maximum absorbance increases with the amount of added silver as well as the con-

centration of modified bases. The more seeds are in solution, i.e. the higher the amount

of modified bases, the more the peak shifts to the longer wavelengths, from 417nm for

the 15-5 oligonucleotides to 430nm for the 30-3 oligonucleotides. For the 30-3 sample the

addition of more silver leads to a red-shift of the peak, for the 15-3 and 15-5 basepair

samples, the peak position seems to be independent of the silver amount. The peaks are

broad compared with the UV-metallized samples, the HWHM values range from 86nm

for short DNA with low modification density to 112nm for long DNA with many modi-

fications. The background, an indication for unspecific silver precipitation, is below 10%

and therefore comparatively low.

The increasing intensity is according to the expectations and indicates larger effective

particle sizes caused by either cluster-cluster-interactions or a coalescence of the single

clusters: The single clusters grown on DNA, that are only separated by few nanometers,

may finally grow together to one particle. The red-shift with increasing modification den-

sity, i.e. seed amount is also understandable: More silver can be deposited, the particles

therefore become larger.

Thus, modified DNA can be metallized by adding a Tollens reagent. The maximum ab-

sorbance depends on the amount of modified bases and increases with time. The samples

are probably not stable for more than one afternoon. The plasmonic peaks are red-shifted

CHAPTER 4. ABSORPTION SPECTROMETRY

Figure 4.19: Temporal evolution of a Plasmon peak for ssDNA in water. The oligonucleotides

are modified 30-3 samples, Tollens with 32mM silver was added.

and broadened in comparison to UV-metallized samples. However, the absorption spec-

troscopy results cannot directly be compared for the UV- and the Tollens-metallized data.

The reason is that two different solutions with different refractive indices, ionic strength,

etc. were used.

4.3.2

Absorption Measurements of Tollens-Metallized ssDNA

The results of double stranded oligonucleotides shall now be compared to measurements

of single stranded oligonucleotides. Single stranded 30-3 DNA was metallized in water and

analyzed by spectrometric methods up to 4hours later. The added silver concentration

was 32mM.

According to expectations, the Tollens metallization also works for single-stranded DNA.

The plasmon peak is red-shifted in comparison to the measurements with dsDNA and

differs in its temporal evolution: Within several hours, the plasmon peak for 30-3 oligonu-

cleotides shifts from 436 to 447nm. The HWHM is above 100nm and therefore in the same

regime as for comparable dsDNA. The maximum absorbance is 62% after 5 hours. This

can be seen in fig. 4.19.

When conducting the experiments with single-stranded DNA in 10mM NaNO

instead

of water, the results hardly differ: The HWHM is even 110nm, the maximum intensity

reaches 80% after 2 hours.

After storing the sample at room temperature overnight, the maximum absorption is

100%, peak wavelength as well as the width of the peak cannot be determined any more.

4.3. ABSORPTION MEASUREMENTS OF TOLLENS-METALLIZED

SAMPLES

The background has also gained intensity. This suggests that the DNA is not stable and

aggregates.

Tollens metallized ssDNA samples therefore hardly differ from metallized dsDNA despite

that the plasmon peak is slightly red-shifted.

4.3.3

Development Solution

Principles

For the reduction of silver in the Tollens reagent, a reducing substance, such as aldehyde

groups, is necessary. Each aldehyde group can only reduce two silver ions, that then form a

negatively charged Ag

cluster[BGM

06]. The excess silver ions in solution are attracted

to these clusters due to electrostatic forces but they cannot be reduced any more. To

achieve a growth of larger clusters, the addition of a development solution (DS) which

reduces the excess silver ions is necessary. The development solution, that I used for my

experiments, consisted of 200µl formaldehyde (35%) and 250µl citric acid (1%) in 100ml

water.

Concentrated Development Solution

20µl of this development solution were added to the samples, where no peak has developed

yet. After some minutes all the samples showed a huge plasmon peak regardless whether

they contained DNA and whether the bases were modified or not. After some hours, for

the sample without DNA the spectra has turned to a broad absorption band without any

peaks, indicating a large number of unspecifically reduced silver clusters sizes, not only

in the nanometer range, present in the solution. The samples containing DNA, however,

show a huge plasmon peak. As the samples were not transmitting any more due to the

strong reaction, the samples had to be diluted by a factor of 20 before measuring. This is

shown in fig. 4.20

There is hardly any difference between the modified and the non-modified DNA. A possible

explanation is, that the development solution has also reduced silver ions, that were

electrostatically attached to the unmodified DNA backbone.

Diluted Development Solution

When adding an amount of 2µl development solution to the samples, only the samples

with modified DNA exhibit a plasmon peak, as shown in fig. 4.21.

The addition of 2µl development solution enhanced the maximum absorption by a fac-

tor of seven (3.2mM Tollens) or four (32mM Tollens) respectively. The addition of DS

slightly increases the HWHM (98nm instead of 91nm). But as the use of development

solution allows for lower silver concentrations, smaller peaks with a HWHM of 74nm can

be produced. The plasmon peak is blue-shifted (from 417 to 410nm).

Duration of the Development Process

For a controlled nanoparticle growth it is necessary to stop the development process

deliberately.

CHAPTER 4. ABSORPTION SPECTROMETRY

Figure 4.20: Absorption two hours after adding 20µl of the development solution.

Figure 4.21: Comparison of absorption spectra with and without development solution

4.3. ABSORPTION MEASUREMENTS OF TOLLENS-METALLIZED

SAMPLES

One idea is to filter out the development solution after a certain time. Thus the sample

was filtered for 30min with a Millipore filter membrane with a pore size of 0.2µm. Taking

a UV/vis spectrum afterward showed that the amount of nitrate ions was significantly

reduced in comparison with the original solution, which is indicated by the steep drop of

absorption below 300nm. The UV/vis spectra for two identically prepared samples, one

with and one without filtering, show almost identical plasmon peaks indicating that the

development process has not been stopped. A slightly higher maximum absorbance for the

filtered sample even indicates that the filtration might accelerate the development process.

This might be due to catalysis effect on the membrane surface. Using concentrators as an

alternative filtering method is impossible due to its long duration that makes investigations

of the temporal evolution impossible.

Another possibility to stop the cluster growth is acidifying the solution as the reduction

potential of silver is pH dependent. Addition of most acids, such as HCl or HSO

leads to

the precipitation of insoluble silver salts. According to [Blu03] the addition of nitric acid

destroys the existing silver clusters by oxidizing the atoms:

Ag + HN O

+ H

O + N O

. Thus an optimum method to stop the development process is still to be found. A promis-

ing method is acidifying the solution with some different chemicals, such as carboxylic

acid.

In conclusion, development solutions enhance the plasmon intensities and therefore make

it possible to work with only few modified bases and low silver concentrations. The chal-

lenge is to control the development process in such a way that it can be stopped again.

4.3.4

Data Analysis and Discussion

Affinity constants and kinetic rates

The experimental results presented in the previous chapters shall now be discussed with

regard to their affinity constants and kinetic rates. The first one addresses to the 'binding

strength' of the silver to the DNA, the second one covers the temporal evolutions.

The peak intensities for the three types of modified oligonucleotides one hour after adding

the Tollens reagent are plotted in fig. 4.22 in dependence of the silver concentration.

The measured data, absorption vs. concentration, are fit with a function

A = Const. ·

1 + cK

according to the Langmuir model, where I is the plasmon peak intensity, c the silver

concentration in M and K

the affinity constant, see [LKSH00] for reference. The inverse

of the affinity 1/K

is the half-saturation concentration, where half of the binding sites are

occupied. One hour after complexation, the half saturation value

is 7mM for the 30-3,

CHAPTER 4. ABSORPTION SPECTROMETRY

Figure 4.22: Concentration dependence of the plasmon peak intensity for different DNA length

and aldehyde density. The spectrum was taken one hour after addition of the Tollens reagent.

The solid lines are the fits described in the text.

5mM for the 15-3 and 3mM for the 15-5 DNA. The average half saturation constant per

modified base is 1mM. As the saturation has probably not been completed after one hour,

the calculated half saturation values might exhibit large errors. The short time span of one

hour was chosen to avoid any influence of processes such as cluster-cluster-agglomeration.

The temporal evolution of the plasmon peak intensity for a silver concentration in the

saturation regime is shown in graph 4.23. The reaction is fast in the beginning and then

slows down. An exact saturation value cannot be extracted from the experimental data

because the clusters aggregate overnight and this falsifies the data. A normalization of

the curves in fig. 4.23 (not presented here) shows that the temporal evolution is fastest

for the 30-3 sample. 15-5 and 15-3 samples have similar development velocities, the 15-3

sample is slightly faster.

Fig. 4.24 shows the temporal evolution of the peak intensity after 2µl of the development

solution have been added. Two samples containing 30-3 oligonucleotides were therefore

kept at room temperature for 90min after 3.2mM and 32mM of Tollens, respectively, had

been added. Then the development solution was added.

The more silver is present in solution, the faster the development solution works. A sat-

uration of the plasmon peak's growth is reached after one to three hours.

4.3. ABSORPTION MEASUREMENTS OF TOLLENS-METALLIZED

SAMPLES

Figure 4.23: Temporal evolution of the plasmon peak intensity for different DNA lengths and

modification densities

Figure 4.24: Temporal evolution of the plasmon peak intensity for two different silver concen-

trations after adding 2µl of a development solution

CHAPTER 4. ABSORPTION SPECTROMETRY

Discussion

Tollens-Metallization exclusively metallizes modified oligonucleotides. With absorption

spectroscopy the growth of nanoparticles can be monitored. The reaction speed as well

as the maximum absorbance depend on the number of modified bases. Addition of a

developing solution speeds up the kinetics and enhances the intensity. There are only

slight differences between metallized ssDNA and metallized dsDNA. A second plasmon

peak could not be found be deconvoluting the data. This might be an indication that the

particles are less rod-like. Another possible explanation is, that the second peak cannot be

resolved due to the large bandwidth: The peaks are broad - the HWHM is about 100nm -

and red-shifted - typical peaks occur at 440nm - compared to UV-metallized samples. To

analyze whether this originates from larger particles sizes or from extrinsic effects such as

the refractive index in solution or the ionic strength, a different measurement technique

must be applied. An optical method to determine particle sizes down to the nanometer

range, Dynamic Light Scattering, will be introduced in the next chapter.

Chapter 5

Dynamic Light Scattering

In the following chapter the principle of Dynamic Light Scattering (DLS) will be explained.

Simulations about the DLS results of rodlike nanoparticles will be presented. Furthermore,

experimental results obtained for UV- as well as for Tollens-metallized samples will be

shown and discussed.

5.1

Principle of Dynamic Light Scattering

Dynamic Light Scattering, DLS, is a time-resolved measurement technique, that provides

information about the size of small particles with diameters ranging from nano- to mi-

crometers.

5.1.1

Measurement Technique

A scheme of the setup for this measurement is the following: A cw laser beam irradiates

the sample at a fixed wavelength that has to be chosen so that the sample shows sufficient

scattering intensity at this wavelength. At a fixed angle, typically 90 degrees, a photode-

tector measures the scattered intensity and sends the signal to an autocorrelator whose

output is processed by a computer program. A scheme of this is shown in fig. 5.1.

5.1.2

Fundamentals

The physical principle of DLS is a decorrelation of the scattered electrical field or the

scattered intensity, respectively, due to Brownian motion of the particles.

When the sample is irradiated, all the particles in the illumination volume act as sec-

ondary sources and scatter the light as explained in 2.2.1. Depending on the inter-particle

CHAPTER 5. DYNAMIC LIGHT SCATTERING

Figure 5.1: Simplified illustration of the DLS setup: A laser beam irradiates a sample at a fixed

wavelength. The scattered intensity is measured by a photodetector and sent to an autocorrelator

Figure 5.2: Random fluctuations of the scattered intensity due to Brownian motion. Only during

a very small time span, the signal is correlated.

distances, the scattered electromagnetic waves of these secondary particles interfere con-

structively as well as destructively and sum up to an effective scattered wave that is seen

by the detector. Since the particles move due to Brownian motion, intensity fluctuations

are measured with respect to the average scattering intensity.

When the signal is measured at two times that are close together, hardly anything will

have changed in the illumination volume and the outcome signals are almost the same.

The correlation for these two measurements is therefore close to 1. When the signal is

measured again after a certain span of time, the particles will have moved in respect

to each other due to Brownian motion. Because of this movement, the phase relations

between the scattered waves have changed and the effective sum signal differs from the

original one. There is no correlation any more between the initial signal and the signal

measured after a certain time span. This principle is sketched in fig. 5.2 and 5.3.

Thus the measurement provides a correlation function as shown in fig. 5.4, defined as

ACF=

t=0

· I(t)dt, that starts at value 1 (=fully correlated) and falls to zero for long

intervals. This decrease is due to the random fluctuations of the detected signal as the

particles undergo random Brownian motion. The Cauchy-Schwartz inequality shows that

the signal at an arbitrary time can never exceed the maximum value which is reached at

the origin of time.

5.1. PRINCIPLE OF DYNAMIC LIGHT SCATTERING

Figure 5.3: Physical principle of the decorrelation of the scattered signal due to Brownian motion:

In short time spans the particles hardy move with respect to each other and thus the effective

output signal remains approximately constant. This is not valid any more for longer time periods,

when the particles have randomly moved: The output signals are not correlated any more.

Figure 5.4: Measured autocorrelation function for a UV-metallized sample (1µM 48mer dsDNA

in 10mM Tris buffer, Ag-to-base 1:1, 10min irradiation)

CHAPTER 5. DYNAMIC LIGHT SCATTERING

From this autocorrelation function one can yield the decorrelation time , that is defined

as the time when the signal has dropped to half of its initial value. The decorrelation time

is inverse proportional to the diffusion coefficient D:

· D

where q is the wave vector of the scattered light that is defined as

q =

sin/2

with the refractive index n, the wavelength and the scattering angle . The diffusion

coefficient is related to the size of the particle by the Stokes-Einstein-equation:

D =

with k

the Boltzmann constant, T the temperature and the viscosity of the solution.

is the hydrodynamic radius of the particle which is assumed to be spherical. For

particles without sharp borders the hydrodynamic radius can differ enormously from the

real radius.

An illustrative example for these equations is the following: When the particle size in-

creases, the motion becomes slower and therefore the diffusion coefficient decreases. We

can see this as an increased decorrelation time in the spectrum.

5.1.3

Polarized Dynamic Light Scattering

More information about the particles can be extracted from polarized measurements: One

linear polarizer is placed between the laser source and the sample and lets only light pass

through that is polarized perpendicular to the scattering plane spanned by the incoming

and the scattered beam. This polarizer is necessary as the laser beam is not completely

polarized from the beginning. A second polarizer - the so called 'analyzer' - is inserted

between the sample and the detector.

For optically inactive spherical particles there is no polarization rotating in scattering

[KV83] meaning that if the two polarizers are perpendicular (so called VH mode), the

detected intensity is zero. This is not the case for elongated particles, such as cylinders or

spheroids, even if they are randomly orientated.

Now two processes are responsible for the decorrelation of the light: The translation of

the particles - as described above - as well as the rotation of the particles. One can

distinguish between two diffusion coefficients, the 'translational diffusion' D

and the

'rotational diffusion' D

, that depend among others on the axis ratio of the particle.

5.2. UNPOLARIZED MEASUREMENTS

5.1.4

Apparatus

First measurements were done with a Triton Hellas Axios 150 instrument (Triton Hellas,

Greece). This setup consists of a diode laser emitting at 658nm with a power of 35mW

that could be attenuated with different filters. A photodiode was positioned to detect the

scattered light at 90 degrees. Between the sample holder and the photodiode an aperture

guaranteed for a narrow laser beam. The autocorrelator used hat a dead time of 11.6ns.

For polarized measurements, linear polarizers of Thorlabs GmbH, Germany, were used. In

the depolarized measurements the incoming beam was not perfectly linearized. The major

drawbacks of this setup are the insufficient laser power, the disability to change the scat-

tering angle as well as a suboptimal software, that did not allow to detect low scattering

intensities. Therefore further measurements were conducted with an ALV-Langen setup

(ALV Vertriebsgesellschaft mbH, Germany) consisting of a 150mW HeNe laser. The angle

could be automatically varied between less than 30

and 155

. Polarizers were already

integrated in this setup.

5.2

Unpolarized Measurements

Several UV- as well as Tollens-metallized oligonucleotide solutions were analyzed by un-

polarized Dynamic Light Scattering. These measurements were intended to give first in-

formation about the particle sizes available in solution.

5.2.1

Experimental Results

for the analysis of DLS data, several methods of varying complexity are available. Two of

them shall be discussed in the following and be applied to the measured data.

Single Exponential Analysis

Assuming a monodisperse sample, the decorrelation is caused by only one correlation

time. The correlation graphs can therefore be fit with single exponential decay functions:

I(t) = f ·

1 +

+ C

I is the intensity measured by the detector, f and C are constants and is the correlation

time (for reference, see e.g. [Ran05], page 153. The fitting procedure was done with the

program Origin that executed a Levenberg-Marquardt iteration. The hydrodynamic radius

can then be calculated as

k · T · q

6 · ·

CHAPTER 5. DYNAMIC LIGHT SCATTERING

(a) 10min UV

(b) 45min UV

Figure 5.5: Measured VV DLS data for a 96bp dsDNA, UV-metallized, with an Ag-to-base ratio

of 1:1. One can see that the deviations from the single exponential decay fit increase with time.

The hydrodynamic radii, that can be calculated from the decay time of the fit function, are in

the range of several tens of nanometers and therefore much larger than expected

UV-Metallized DNA

UV-Metallized samples with a silver-to-base ratio of 1:1 and various lengths and irradia-

tion times were investigated in the Triton-Hellas setup, the data was analyzed by single

exponential analysis.

The resulting hydrodynamic radii range from 33nm for 23mer DNA that was irradiated

for 10min up to 46nm for 96mer DNA, also irradiated for 10min. When increasing the ir-

radiation time from 10 to 45min, the radius for the 96mer DNA remains almost constant,

whereas the radius for 23mer DNA increased to 56.5nm. The deviation of the measured

data from a single exponential fit function increases with irradiation time. Typical mea-

sured data as well as the single exponential decay fits are shown in fig. 5.5.

Both parameters varied, the DNA length as well as the irradiation time, seem to have

an influence on the hydrodynamic radius. Comparing the outcome with the UV/vis spec-

trometry results that are given in 4.2.7, it can be seen that the particle sizes are much

larger than expected. However, it is known that large particles contribute much stronger

to the scattered intensity (d

) than small particles. Therefore, the influence of a few large

particles which are present in solution might dominate over the contribution from small

particles. Hence, a more complex analysis method, that accounts for more than one par-

ticle size, is necessary.

Tollens-Metallized DNA

Several Tollens-metallized DNA samples of various lengths, modification densities and

silver concentrations were prepared and stored overnight before the light scattering was

measured and analyzed by the single exponential decay method.

The measurement, done with the Triton-Hellas-setup, showed significant scattering in-

5.2. UNPOLARIZED MEASUREMENTS

Figure 5.6: VV DLS data of a Tollens-metallized 30-3 sample, 1.5mM Ag, stored overnight. A

single exponential decay fit yields a hydrodynamic radius of several tens of nanometers which is

much higher than expected.

tensity only for medium silver concentrations between about 0.32mM and 16mM, also

depending on the DNA length. The hydrodynamic radii calculated from the fit single ex-

ponential decay, range from 56nm to 104nm without any observable tendency. One typical

spectrum as well as the single exponential decay fit is shown in fig. 5.6.

These results can be explained as follows: For silver concentrations below 0.32mM the

produced particles are too small to scatter light efficiently. For silver concentrations above

16mM, the particles might have aggregated to particles larger than 200nm. These particles,

however, are filtered out before the measurement. Thus, a sufficient scattering intensity

is only reached for an intermediate silver concentration. Thus the results achieved by

single exponential analysis of the DLS data support the spectrometric result that Tollens

metallized samples aggregate overnight. The results for the radii are of the same order of

magnitude as the single exponential decay results for UV-metallized samples. They are,

however, roughly a factor of two larger. This agrees with the spectrometric results, where

plasmon peaks of Tollens-metallized samples were red-shifted compared to UV-metallized

samples, corresponding to an increase in size.

Like for the UV-metallized samples, the hydrodynamic radii for Tollens-metallized samples

are, however, much larger than expected. Therefore a more sophisticated analysis method

must be applied to account for effects like polydispersity and various distinct particle

sizes. This is explained in the next paragraphs.

5.2.2

CONTIN Analysis

The previous analysis has shown, that single exponential decay analysis of the results

is not sufficient. In principle, it is possible to extend the fit to include a sum of several

exponents, but this makes the fit unstable. Furthermore, the width of the size distributions

CHAPTER 5. DYNAMIC LIGHT SCATTERING

cannot be analyzed by this simple method. Therefore a commercial DLS analysis program

of ALV, Germany, was used.

For measurements done with the ALV setup, a CONTIN analysis of the particle sizes is

possible. The CONTIN algorithm, that was invented by S. Provencher [Pro82], uses an

inverse Laplace transformation for determining the particle sizes. For the calculated size

distributions, three different types of weighting can be chosen: The unweighted signal, also

known as intensity-weighted signal, is proportional to d

, according to Rayleigh, as shown

in 2.2.1. In the mass-weighted spectrum, the intensity is proportional to d

. This weighting

type is important for comparing the DLS data with the UV/vis data, as the plasmonic

absorption intensities of nanoparticles are also proportional to d

in a first approximation.

The number-weighted spectrum shows the number of particles in solution. All spectra are

normalized to the peak of maximum intensity.

UV-metallized samples of various parameters were freshly prepared and investigated with

DLS. The CONTIN analysis detected up to three or four particle sizes: A 23mer DNA

sample with an Ag-to-base ratio of 2:1 and 10min irradiation shows peaks at 0.5nm, 1-

2nm, 8nm and a small but broad distribution for larger particle radii. The normalized,

mass-weighted spectrum can be seen in fig. 5.7a), the number weighted spectrum can be

seen in 5.7b).

When the irradiation time is increased to 90min the peak below 1nm shifts to larger radii

as shown in fig. 5.8. The amount of particles having radii larger than 10nm, however,

increases.

Increasing the silver amount to a silver-to-base ratio of 20:1 increases also the particle

sizes, as shown in 5.9. The amount of particles having radii larger than 10nm, however,

increases. The 0.5nm particles increase to 0.7nm, the 1-2nm particles grow to a radius of

2-3nm. The peak around 10nm vanishes completely, another peak occurs at 40nm. The

intensities of larger particles have significantly decreased in comparison to the peak below

1nm. The second-smallest peak has become much narrower.

When longer oligonucleotides with 96 instead of 23 basepairs are metallized, the average

radius of particles smaller than 1nm shifts to 0.7 and drastically loses intensity, whereas

the 1-2nm peak has gained intensity. Moreover, peaks at 5nm and 30nm appear. This is

shown in fig. 5.10

Experiments with an unbuffered solution were done with 48mer DNA. The DNA with

a silver-to-base ratio of 10:1 was metallized in an aqueous solution with 10mM NaNO

at pH 8 - and afterward examined with DLS. The result of a CONTIN analysis can be

seen in fig. 5.11. Due to a different DNA length the CONTIN results cannot be directly

compared to one of the samples metallized in 10mM Tris buffer. In the unbuffered sample

no peak occurs for radii below 1nm - such a component exists, however for 23bp DNA with

a Ag-to-base ratio 20:1 as well as for the 96bp sample. The unbuffered sample exhibits

two more particle radii, at 9nm and at 50nm. These peaks are red-shifted compared to

the samples metallized in Tris buffer. This increase in particle size is in accordance with

UV/vis absorption spectra: When DNA was metallized in an unbuffered solution, the

5.2. UNPOLARIZED MEASUREMENTS

(a) mass-weighted spectrum

(b) number-weighted spectrum

Figure 5.7: Mass-weighted and number-weighted size distributions of metallized 23mer DNA

with a silver-to-base ratio of 2:1 and 10min irradiation

CHAPTER 5. DYNAMIC LIGHT SCATTERING

Figure 5.8: Comparison of mass weighted size distributions of metallized 23mer DNA with a

silver-to-base ratio of 2:1 and 10min and 90min irradiation

Figure 5.9: Comparison of mass weighted size distributions of metallized 23mer DNA with silver-

to-base ratio of 2:1 and 20:1 and 10min irradiation.

5.2. UNPOLARIZED MEASUREMENTS

Figure 5.10: Comparison of mass weighted size distributions of metallized 23mer and 96mer

DNA

Figure 5.11: Mass-weighted size distribution in an unbuffered NaNO

solution at pH 8 containing

metallized DNA. Three particle sizes are available in the sample.

CHAPTER 5. DYNAMIC LIGHT SCATTERING

plasmon peak shifted to long wavelengths indicating an increase in size. The assumption,

that this is accompanied by an increase in the size distribution, indicated by a broad

peak in the UV/vis spectrum, is not confirmed by the DLS data: The widths of the size

distributions is similar to the Tris buffer samples.

All spectra discussed were recorded at 90

. Varying the angle between 30

and 155

changed the results only slightly.

The CONTIN analysis showed that at least three different particle sizes are present in

solution, whereby the smaller ones are the most frequent. The particle sizes broaden and

shift to bigger radii when the irradiation time is prolonged. This is in accordance with

absorption spectrometry results that the photoinduced reduction is not finished before

60min. In that time longer irradiation time means more silver deposition (and thereby

decreasing the axis ratio of the prolate particles). A larger amount of silver ions induces a

slight shift to larger radii and significantly narrows the spectrum. A possible explanation

is that the silver saturation value has still not be reached as it is also indicated by the

absorption spectrometry results: If it is further assumed that photoinduced silver deposi-

tion is a self-limited process leading to stable nanoclusters, more silver means that more

clusters with the final size can be built. This agrees with the fact that the second smallest

peak has not only been narrowed but also shifted to a larger radius. Using longer DNA,

the smallest particle size becomes less frequent, whereas the other peaks gain intensity.

This meets the expectations that larger DNA come along with larger nanoparticles.

5.2.3

Temporal Evolution

DLS measurements can be used to monitor the growth of nanoparticles with the Tollens

method. A sample containing 1µM of 15-3 DNA modifications was prepared and filtered

and 16mM silver in a Tollens reagent were added. Every five minutes DLS a spectrum

was taken with the ALV setup. They are shown in fig. 5.13

The spectra show a large-radius contribution that can be attributed to unidentified back-

ground in the unfiltered Tollens solution and that provides for enough scattering intensity

to record correlation graphs. After 5min a very small contribution of 10 to 20nm particles

develops. Five more minutes later, the peak intensity has increased and the asymmetric

peak has shift to 10nm. 15min after addition of the Tollens aliquot the peak position is

at 7nm. In the following time it fluctuates between 3 and 8nm. Presumably, these fluc-

tuations reflect the limited measurement accuracy. The scattering intensity increases by

a factor of two during the growth process. Fig. 5.12 shows the development of the peak

intensity and the radius with time. The CONTIN analyzed spectra of the growth process

are shown in fig. 5.13.

In comparison to the UV-metallized samples, only one peak size below 100nm is present in

the solution. The width of the size distribution is much larger than for the UV-metalized

sample, it ranges from 2nm up to several tens of nanometers. This is in accordance with

the broad plasmon peaks in the absorption spectra.

5.2. UNPOLARIZED MEASUREMENTS

Figure 5.12: Temporal evolution of the peak intensity and the peak position for 15-3 DNA to

which 16mM of silver in a Tollens reagent were added

In conclusion, DLS measurements can be used to monitor the growth of particles in

solution. Already 5min after addition of the Tollens reagent first clusters have been built.

The particle size decreases with time. One possible explanation is that ions and silver

diamine complexes in solution are electrostatically attracted to the negatively charged

DNA backbone and increase its hydrodynamic radius. The more silver is reduced, the

more compensated the charge of the DNA is and therefore less particles are attracted: the

large diffuse ionic shell is replaced by smaller silver clusters. Silver clusters scatter visible

light efficiently, this makes the intensity increase. Further measurements have to be done

to confirm or confute this argument.

5.2.4

Reference Samples

A reference solution containing just Tollens but no DNA exhibited almost the same scat-

tering intensity as the sample with DNA. Particle sizes of 200nm and 2µm were also

present in this sample but smaller particles did not develop within 60 minutes.

A solution containing only DNA without silver clusters does not scatter at all in the

Triton-Hellas setup, even when its concentration is increased by a factor 50 to 50µM.

Triton-Hellas measurements on freshly prepared Tollens-metallized samples do not provide

enough scattering intensity for analysis.

The Triton-Hellas setup could not detect scattering of irradiated reference samples con-

CHAPTER 5. DYNAMIC LIGHT SCATTERING

Figure 5.13: Temporal evolution of nanoparticles during Tollens-metallization of 15-3 DNA to

which 16mM of silver in a Tollens reagent were added

taining buffer solution and silver but no DNA. The more sensitive ALV setup could not

detect more than about 3 counts per second (cps), which is three times the background

scattering signal. This small signal can be attributed to particles with a radius of 30 to

40nm, probably unspecified silver deposition due to dirt in the solution. Smaller particles,

as they were present in all metallized DNA samples, could not be detected. For Tollens

reference samples the situation is more complicated: A fresh undiluted Tollens solution

does not scatter when analyzed by the Triton-Hellas setup. When ammonium evaporates

from the solution, the silver diamine complexes are dissolved and unsoluble silver oxide

falls out again. These silver oxide particles are able to scatter light. In samples used for

metallization this effect is less important as the Tollens solution is diluted by a factor

10 to 100. This problem can be easily overcome by preparing the Tollens reagent freshly

before the measurement.

Sensitivity of the apparatus

Silver spheres with a radius of 10nm ±2.5nm were provided by BBI/Tedpella at a con-

centration of 1.2nM. Without any filter, the scattering intensity was 7 to 11cps for the

Triton-Hellas- and 20cps for the ALV-setup at a laser attenuation of roughly 50%. A

CONTIN analyzed spectrum can be found in the appendix B; it shows that the evaluated

diameter corresponds to the expected one. At the Triton-Hellas setup an intensity of 7cps

or larger is necessary to record data, at the ALV-setup 1cps is sufficient.

5.3. POLARIZED MEASUREMENTS

5.3

Polarized Measurements

For polarized measurements the theory is more complex and there is no widely accepted

algorithm to calculate the dimensions of the nanorods. Thus I have performed simulations

with the program Matlab: Using the formulas described below I calculated the correlation

times for various axes' lengths for both orientations of the polarizers. The experimental

values can then be compared to the theoretical ones and the length and diameter can be

determined in principle.

5.3.1

Simulations

Garcia de la Torre and Tirado developed formulas for the translational and rotational

diffusion coefficients of prolate ellipsoids [TG80]:

(log(a/b) + 0.312 + 0.565 · (b/a) + 0.100 · (b/a)

)

(log(a/b) - 0.662 + 0.917 · (b/a) - 0.05 · (b/a)

)

whereas a is the long and b the short axis of the ellipsoid. D

has the unit m

/s, D

given in 1/s.

The prefactors show that the order of magnitude of the coefficients as well as the ratio

mainly depends on the length of the long axis a.

Van der Zande [vdZDBP00] suggests and performs DLS measurements with parallel

polarizers (so called vertical-vertical -VV- mode) and with crossed polarizers (vertical-

horizontal -VH- mode). According to van der Zande, the diffusion coefficients are related

to the decay times as follows:

-1

V V

= 2q

-1

V H

= 2q

+ 12D

For data acquired by the Triton-Hellas software, the right sides of these equations have

to be divided by 2 as the decorrelation of the electric field was analyzed instead of that

of the intensity. With these formulas for the electric field decorrelation I calculated the

diffusion coefficients in dependence of the long axis a and the short axis b in Matlab. The

result is shown in fig. 5.14 und 5.15.

Some general trends can be seen from the formulas above: The longer the particle, the

smaller the difference between

V V

and

V H

and the larger are

V V

and

V H

. The longer

CHAPTER 5. DYNAMIC LIGHT SCATTERING

Figure 5.14: Calculated correlation time in the VV mode in sec. The x and the y-axis show the

long and the short axis of the ellipsoid in m.

Figure 5.15: Calculated correlation time in the VH mode in sec. The x and the y-axis show the

long and the short axis of the ellipsoid in m.

5.3. POLARIZED MEASUREMENTS

(a) VV mode

(b) VH mode

Figure 5.16: Correlation time in the VV and VH mode dependent on the axis ratio. A particle

length of 10nm was assumed

the short axis b, the bigger the correlation times are as shown in fig. 5.16. The steepness

of this increase is different for the two modi, however.

Fig. and 5.16 shows the correlation time for different axis ratios. Therefore a particle

length of 10nm was assumed. The correlation time in the VH mode is more than two

orders of magnitude smaller than that of the VV mode.

5.3.2

Experimental Results

Only aspherical particles should possess a polarized contribution, for spherical particles

the scattering intensity for crossed polarizers should be zero. This expectation was checked

with commercial spherical Latex spheres with a radius of 40nm: The signal in the VV

mode is at least a factor 175 more intense than the signal in the VH mode, that is only

marginally above the background signal.

The metallized DNA samples, however have a significant polarized contribution, the VV

and the VH signals differ by a factor of 4 to 8. This indicates significantly aspherical

particles.

Single and Double Exponential Fits

To get a first idea about the decay times, the spectra were again analyzed in Origin. This

time also a double exponential decay is applied.

Single exponential fits for samples with UV-metallized DNA of various lengths provided

correlation times

V H

around 20µsec for 10min UV irradiation. As this value is compara-

tively high and differs from the VV-time by less then one order of magnitude, the results

cannot be quantitatively compared with the theoretical calculations. For the depolarized

measurements a CONTIN analysis has pointed out the fact that two particle sizes are

CHAPTER 5. DYNAMIC LIGHT SCATTERING

(a) 10min UV

(b) 45min UV

Figure 5.17: Measured VH DLS data for a 96bp dsDNA, UV-metallized, with an Ag-to-base ratio

of 1:1. One can see that the deviations from the single exponential decay fit increase with time.

For 45min irradiation two distinct correlation times can be calculated with a double exponential

fit function.

present in solution. Thus a single exponential decay function is not appropriate although

it matches well at first sight.

The VH-spectra of samples irradiated for 45min show a curve shape that deviates from

a single exponential decay. A double exponential decay function shows two correlation

times, that are separated by at least one order of magnitude: 23mer DNA has 180µs

and 7.1µs, 96mer DNA has 450µs and 20µs. These two correlation modes, both in the

VH-mode, indicate that at least two particle sizes are available in solution.

Typical VH-spectra for 10min and 45min irradiation time as well as their exponential fits

can be seen in fig. 5.17.

For Tollens metallized DNA stored overnight, the VH correlation times are between 88µs

and 130µs for single exponential analysis and therefore exceed the UV-values again. Fig.

5.18 shows typical measured data as well as the single exponential decay fit.

This simple analysis showed the presence of an aspherical term. Again, the calculated

decay times are much too long. For quantitative information, a CONTIN analysis is

necessary.

CONTIN Analysis

UV-metallized samples were measured with DLS in the VV and the VH mode. The CON-

TIN analysis of the decay times in both modes is shown in fig. 5.19.

For the 23mer and the 96mer with low silver concentrations and short irradiation time,

there is a dominant peak at 390µs in the VV mode. In the VH mode a peak at 17µs for the

23mer and 38µs for the 96mer becomes more dominant. The peaks in the VH mode are

broadened. For the 23mer a peak at 1.2µs, for the 96mer a peak at 4.2µs occurs in the VH

5.3. POLARIZED MEASUREMENTS

Figure 5.18: VH DLS data of a Tollens-metallized 30-3 sample, 1.5mM Ag, stored overnight.

The time-constant yielded is 2.3 times faster than in the VV mode.

mode. When the irradiation time is longer, the peaks in the VH mode are also broadened:

Only one peak at 300µs - instead of 380µs in the VV mode - and some shoulders are

distinguishable. For the sample with larger amount of silver the long decay time does

not depend on the mode. The peaks at shorter decay times, however, are broadened and

shifted to shorter times. A short decorrelation time, 0.2-0.3µs occurs. The intensity of the

short time peaks is enhanced in comparison to the VV spectrum.

The CONTIN analysis showed non-vanishing VH spectra indicating aspherical particles.

At least three different decorrelation times can be read off the VH spectra correspond-

ing to various particle sizes or shapes. Compared to VV spectra, the intensity of short

decorrelation times is enhanced; this is in accordance with the results of the simulations.

Before the data can be compared to the simulations and before conclusions can be drawn,

the peaks of one particle size in the VV and VH mode must be correctly to each other.

To do so, measurements with HPLC

purified samples have to be done.

High Performance Liquid Chromatography

CHAPTER 5. DYNAMIC LIGHT SCATTERING

(a) 23mer, Ag-base 2:1, 10min UV,

(b) 23mer, Ag-base 2:1, 90min UV

(d) 96mer, Ag-base, 1:2, 10min UV

Figure 5.19: CONTIN analysis of the VV and VH decay times for various UV-metallized samples.

Chapter 6

Stabilization of the Nanoparticles

This chapter presents the fundamentals of nanoparticle stabilization and presents first

results achieved with two different stabilizers.

6.1

Fundamentals

In nanoparticle synthesis it is very important to stabilize the colloids against aggregation

due to attractive forces, e.g. van der Waals forces. This is especially valid for particles

made of silver, as its tendency for aggregation is larger than for many other metals, such

as gold. The stabilization can be realized either sterically or electrostatically.

For steric stabilization polymers such as PVP

are adsorbed or chemically bound to

the surface of the nanoparticle, as shown in fig. 6.1. Because of these organic shells the

Figure 6.1: Scheme of steric stabilization: The nanoparticles are covered by a shell of polymers.

particles cannot get close enough to be attracted by the short ranged van der Waals forces.

Polyvinylpyrrolidone, for the experiments done in this work polymers with an average weight of

10.000 Dalton, provided by Sigma-Aldrich, Germany, were used

CHAPTER 6. STABILIZATION OF THE NANOPARTICLES

When polymers, which are soluble in the solution, are used, no aggregation occurs among

the covered nanoparticles. A drawback of this method is, however, that the polymers,

that have weights of more than 10.000 Dalton

and are tens to hundreds of nanometer

long, immensely increase the hydrodynamic radius; this makes it difficult to identify the

'real' particle diameter by techniques like, for instance, DLS. According to [Blu03] organic

substances, i.e. PVP in this case, act as inhibitors for crystallization as they bind to the

growing clusters. Therefore the final size of clusters is reduced whereas the number of

clusters is increased.

In the case of electrostatic stabilization, the surfaces of the nanoparticles are charged.

These surface charges yield an arrangement of ions in solution around the particle to

build up a counter-field. A scheme of this is shown in fig. 6.2: The repulsive Coulombic

Figure 6.2: Electrostatic stabilization: The nanoparticles carry a surface charge, here drawn as

negative, counter ions in solution arrange around the particles and partly screen the charges.

The Coulomb forces among the particles prevent the particles from agglomerating.

forces stabilize the particles. The advantage of this stabilization method is, that it does

not change the radius of the DNA. One drawback is its sensitivity to the pH value in

the solution as well as the ionic strength. Most frequently sodium citrate is used as an

electrostatic stabilizer: This substance also acts as a reducing agent for silver ions. Therefor

synthesis conditions must be carefully chosen so that the clusters really grow on the DNA

pattern instead of being reduced homogeneously in solution.

6.2

Experimental Results

Measurements with platinum done by Mertig and Pompe [MP04] showed that a stabiliza-

tion of the metallized DNA is not necessary as no aggregation occurs. In papers dealing

with silver-DNA-structures, stabilization of the particles is simply not mentioned. The

authors typically just write, that clusters were heterogeneously grown on DNA while

homogeneous deposition in the surrounding medium is suppressed.

The charge of my DNA-silver-structures and thus their intrinsic electrostatic stabilization

of the nanostructures is unknown. DLS data indicate small, probably not agglomerated,

1 Dalton = 1 atomic mass unit = 1.66E-27 kg

6.2. EXPERIMENTAL RESULTS

particles as well as larger ones. AFM micrographs, presented in chapter 8, show linearly

agglomerated structures. It is not known, whether the particles already agglomerate in

solution or when being brought on substrate. UV/vis data do not allow for quantita-

tive information about the particle size. The large bandwidth of the plasmonic peaks of

Tollens-metallized samples indicate a very broad size distribution, the temporal evolution

of these peaks implies that the solutions are not stable for more than some hours. Thus a

stabilizer is recommendable. First experiments were done with the UV-metallized samples

as the measurement conditions are better controllable in this case. PVP with an average

molecular weight 10.000 Dalton and sodium citrate (all provided by Sigma-Aldrich, Ger-

many) were used as stabilizers.

When adding the stabilizers to the sample before the UV-irradiation, plasmon peaks at

414nm (sodium citrate) or 425nm (PVP), respectively occur even if no DNA is present in

solution. This is because of the reducing character of the two stabilizers. This problem does

not occur when the substances are added directly after the irradiation. The corresponding

spectra for these reference measurements are shown in the appendix B.

Measurements were carried out in aqueous solutions containing 5mM NaNO

, that were

titrated to pH 8.3 by NaOH. The sample contained 1µM 48mer dsDNA. This solution was

chosen as pre-measurements without stabilizers showed broad peaks at long wavelengths.

Thus the effect of a stabilizer might pronounced as for Tris buffer solutions with narrow

peaks at short wavelengths. The final concentration of sodium citrate in the solution was

0.01mM. PVP was used in a concentration of 5.1mg/l.

Fig. 6.3 shows the plasmon peaks for three different samples: One was unstabilized, to the

others sodium citrate or PVP, respectively were added after irradiation. The amount of

silver was 1mM, corresponding to a silver-to-base ratio of 10:1, the irradiation time was

10min.

For the unstabilized sample a plasmon peak at 454nm occurs. For the sodium-citrate-

stabilized sample, this peak has shifted to 437nm, for the PVP-stabilized sample it has

only slightly shift to 454nm. The unstabilized sample shows the least, the sodium-citrate-

stabilized sample shows the most intense peak. All spectra are so broad, that the band-

width cannot be determined. Comparing the peak shapes in the range below 700nm pro-

vides that the unstabilized and the sodium citrate stabilized samples have almost the same

shape, whereas the PVP-stabilized sample has a broader peak. At 350nm a quadrupole

shoulder can be seen, this is less pronounced for the sodium-citrate stabilized sample.

The reference samples shown in B and discussed above showed that also in presence of

stabilizers the nanoparticles are grown on DNA. The high peak intensities for the stabilized

samples indicate that this growth process is enhanced in the presence of sodium citrate

or PVP. The fact that the spectra change when stabilizers are added confirm that these

substances do interact with the silver nanoparticles. The blue shift as well as the less

pronounced quadrupole shoulder indicate that smaller particles are grown in presence

of sodium citrate. No such effect is seen for PVP. The red-shift can be attributed to a

raised refractive index around the particles due to the organic shell. As the spectrum even

broadens, indicating either larger particles or more polydispersity, no more experiments

CHAPTER 6. STABILIZATION OF THE NANOPARTICLES

Figure 6.3: Plasmonic peaks for unstabilized and stabilized samples. When sodium citrate is used

as a stabilizer the peak is shifted to the blue and the quadrupole shoulder is less pronounced.

PVP does not have any improvement compared with the unstabilized sample. The measurement

was carried out in a non-buffered alkaline aqueous solution, details can be found in the text.

were carried out with PVP. Further measurements concentrated on sodium citrate as a

stabilizer.

For DLS analysis 23mer DNA was metallized, whereby the silver-to-base ratio was 2:1, the

irradiation time was again 10min. For this measurement, again 10mM Tris buffer was used

to provide a better comparison with the samples discussed in 5.2.2. The mass-weighted

as well as the number-weighted CONTIN analyzed spectra are shown in fig. 6.4.

The 0.7nm peak and the 2nm peak in the mass-weighted spectrum of non-stabilized DNA

have merged to one broad peak. The 8nm has shifted to 20nm. The number-weighted

spectrum also shows one broad peak instead of two. This indicates that in Tris buffer the

sizes cannot be reduced sodium citrate. The size distribution even broadens. Although

there is no improvement for the particle size and distribution in this case, this experiments

show that sodium citrate interacts with the metallized DNA and influences the particle

size distributions.

Thus sodium citrate might be nevertheless a working stabilizer for the non buffered solu-

tion of the Tollens-metallized samples. The UV/vis data of the metallization in alkaline

water solution confirms that assumption. As sodium citrate can also act as a reducing

agent, the development solution containing formaldehyde might be replaced by one con-

taining sodium citrate. Sodium citrate will then act as a developer as well as a stabilizer.

More experiments have to be done.

6.2. EXPERIMENTAL RESULTS

(a) mass-weighted spectrum

(b) number-weighted spectrum

Figure 6.4: CONTIN analysis for citrate stabilized 23mer DNA, Ag-to-base ratio 2:1, 10min irra-

diation time. The mass-weighted and the number-weighted spectrum are shown. For comparison

the results for unstabilized metallized DNA are also included.

Chapter 7

Nanorings

The previous chapters deal with metallic nanospheres (from BBI/Tedpella) and nanorods

(metallized dsDNA). But the recognition properties of DNA allow for even more complex

particle shapes.

Figure 7.1: Base sequence of Molecular Beacons [Zuk03]: The complementary parts of the stem

hybridize while the intermediate sequence forms a loop-structure.

Nanorings can be produced by metallizing molecular beacons. These are single DNA

strands with a special base sequence that forms a stem loop structure [TK96] as shown

in fig. 7.1 [Zuk03]: One end of the single strand is complementary to the other one and

therefore the two ends hybridize while the strand sequence in between is 'bent' and forms

a loop.

Hybridization

To produce ring-like structures the stem must have hybridized.

The molecular beacons were labeled with the fluorophore Cy3 at one end. This makes it

possible to observe the hybridization status

: When the stem hybridizes, the fluorescence

increases by approximately 70%. This is a special feature of the used dye and linker system

and has been characterized previously [Ran05]. Thus the fluorescence of molecular bea-

cons in 10mM Tris buffer/50mM NaCl was measured in dependence of the temperature

(not shown here). The main contribution to the temperature dependence comes from the

intrinsic temperature-dependence of the fluorophore, whose signal decreases with temper-

ature. A kink in the curve indicates the melting temperature. The experimental values

63.6

C for the 50mer and 60

C for the 80mer differ by about 13

C from the theoretical

values 77

C for the 50mer and 73.7

for the 80mer calculated by M. Zuker's program

Mfold [Zuk03]. This might be due to the special experiment conditions, such as the buffer

or the concentration. As the effect is overlapped by the fluorophores intrinsic temperature

dependence, errors in evaluation of the values might also play a role. Nevertheless, theory

as well as experiments predict a stem-loop form for the used DNA at room temperature.

According to literature [MKT03] the intramolecular hybridization strength is larger than

the intermolecular one. Therefore the molecular beacon DNA prefers to assemble into

rings instead of hybridizing with a second strand to form a linear molecule.

Thus, molecular beacons in 10mM Tris-buffer/50mM NaCl are hybridized at room tem-

perature and form ring-like structures.

UV-Metallization

The UV-metallization was conducted in the same way as for the linear DNA. UV/vis

spectra showed a plasmon peak, see fig. 7.2.

For the 50bp molecular beacon a symmetric peak with FWHM of 108nm occurred at

412nm. The 80bp molecular beacon showed a 114nm broad peak at 416nm. The red shift

indicates larger particles, as it is expected for the larger molecule.

The Cy3 absorption peaks at 522nm and 557nm vanish during metallization, even before

the nanoparticle peak has developed. At the same time, the color of the solution changes

from red to yellow, indicating that the Cy3 fluorophore reacted with the added Ag. The

immense decrease of the base absorption at 260nm proves that silver is also deposited on

the DNA itself.

Unpolarized DLS measurements, analyzed with CONTIN as shown in fig. 7.3

result in three different particle radii, 2nm, 7nm and 40nm with the relative (mass

weighted) intensities 10:5:1. A low VV:VH intensity ratio of 5.4 points at asymmetric

particle shapes. This asymmetry in the ring-like particles arise from the stem-part of the

The hypochromism method is not applicable here: Because of the short stem length, the difference

in absorption between double and single stranded DNA is hardly detectable

CHAPTER 7. NANORINGS

molecule. The polarized data, however, differ significantly from that of comparable linear

metallized DNA: Only two correlation times, 10

-4

s and 10

-6

s, can be detected and they

are both extremely broads.

As a conclusion, the applied UV-metallization method also works for more complex DNA

structures. More measurements have to be done to yield information about the shape of

the metallized particles.

(a) 50mer

(b) 80mer

Figure 7.2: UV-Metallization spectra of a 50bp and a 80bp long molecular beacon for two

different silver concentrations and 10min irradiation time. Note: The concentration of DNA and

silver was only one fifth of the concentrations typically used for UV-metallization of linear DNA.

CHAPTER 7. NANORINGS

(a) Particle sizes

(b) Decay times

Figure 7.3: CONTIN analysis of DLS data yielded from UV-metallized 80bases ringlike DNA.

a) Comparison between ringlike and linear DNA b) Correlation times of ring-DNA in the VV

and the VH mode

Chapter 8

Atomic Force Microscopy

8.1

Fundamentals

Atomic Force Microscopy (AFM) is a technique to analyze the topography of areas up to

several micrometers. It is possible to visualize structure that have dimensions of only a

few nanometers.

For the investigations the tapping mode was used: A cantilever containing a silicon tip with

a radius of 10nm ('normal tip') or 2nm ('sharp tip') at its end, oscillates at roughly 300kHz,

a frequency near its resonance frequency, with a certain, fixed amplitude. As interactions

with the surface damp the amplitude, a control loop has to correct the distance between

the cantilever and the surface to provide for the constancy. The required readjustments

give information about the distance between the surface and the tip. Scanning of the

surface with the help of a piezoelectric crystal provides the surface topology. The principle

of AFM measurements is shown in fig. 8.1:

Important parameters to adjust are the proportional and integral gain, that regulate the

control loop, the amplitude setpoint, which determines the oscillation amplitude, and the

scan velocity.

Atomic Force Micrographs were taken with a Multimode and Dimension V instrument

(Digital Instruments Inc., Santa Barbara, USA). Tips were provided by Budget Sensors.

8.2

Sample Preparation

An essential point of AFM is sample preparation. As one of the requirements is a good flat-

ness of the surface, silicon wafers were used as substrates. There were two main challenges

for bringing the metallized DNA from solution to substrate:

CHAPTER 8. ATOMIC FORCE MICROSCOPY

Figure 8.1: Schematic of the working principle of an AFM from [Lin06]. The cantilever oscillates

while the surface is scanned with a piezoelectric crystal. Forces between the tip atoms and the

surface atoms change the cantilever's amplitude. A feedback loop compensates for these changes.

Its regulations give information about the surface topography.

8.2.1

Requirements

Salt Removal

The first challenge is the removal of salt from solution. As the salt concentration (typi-

cally more than 60mM) exceeded the DNA concentration by a factor of 60000, it would be

otherwise difficult to find and distinguish the DNA between the large salt crystals. Scan-

ning Electron Microscopy (SEM) images of metallized DNA deposited on a gold surface

without removing the salt can be seen in fig. 8.2.

Due to several reasons discussed in the previous chapters, the DNA metallization has to

be carried out in solutions containing at least some mM of salt. The salt has therefore to

be removed afterward. For this purpose several methods were applied: One was filtering

the solution before dispersing it on surface. This can be done with concentrators provided

by Millipore. These devices, designed mainly for buffer-exchange work as follows: The

solution is filled into a vial that is separated from a larger reservoir by a vertical mem-

brane, whose pore size has to be chosen in advance according to the molecular weight

of the metallized DNA. This concentrator device is centrifugated at 13.000 g for 45min.

Meanwhile the water with the salt molecules is pushed through the membrane. Due to its

size the metallized DNA cannot pass the membrane. A very small rest volume of about

30µl containing the metallized DNA remains. When diluting this with distilled water to

the initial DNA concentration, the salt concentration has decreased. After repeating this

procedure typically five times, the salt concentration was lowered by a factor of 100 000.

The advantage of this method is that the salt concentration can be reduced at will by

repeating the concentration cycles. The drawbacks are that the method is expensive as

well as time-consuming and that there is a risk of agglomeration of the DNA clusters when

they are close together in a small volume in absence of salt. A much faster method of

filtering is to use filter membranes provided by Millipore. This 25mm diameter membrane

8.2. SAMPLE PREPARATION

(a) SEM image of salt crystals

(b) Zoom in SEM image of salt crystals

Figure 8.2: SEM images of 2µl of a solution of metallized DNA on a gold surface without removal

of the salt: The surface is dominated by large salt crystals. The sample consists of an aqueous

solution with 10mM NaNO

0.2µM 48bp dsDNA, 1mM AgNO

. The irradiation time was 45min.

These micrographs indicate the necessity for salt removal.

with pore sizes of 200µm floats on the surface of a water reservoir. Droplets of the solution

with a volume of about 10µl are distributed on the membrane. Due to osmotic pressure

the salt molecules pass the membrane and diffuse into the much larger water reservoir. Be-

cause of their much larger size, the metallic DNA particles remain mostly in the droplets.

After about 25min the droplets are taken off the membrane again using pipettes. This

method is much faster than the concentrator centrifugation. But the main disadvantages

are, that the final salt concentration remains unknown and there is a higher risk to lose

the metallized DNA because of a larger pore size compared to the concentrators.

First dispersing the solution on a silicon wafer and then rinsing it with ultrapure water is

not applicable as the attractive forces between the metallized DNA and the silicon surface

are so weak, that the metallized DNA is also washed off.

Homogeneous Dispersion

The second challenge is to avoid agglomeration: First measurements using commercially

available silver colloids of BBI/Tedpella showed that silver particles have a strong ten-

dency to form large three-dimensional clusters as can be seen in Scanning Electron Mi-

croscopy (SEM) images like in fig. 8.3.

Experiments with the BBI silver spheres also indicated that simply diluting the solution

does not solve the problem as it only reduces the number of colloidal aggregates but not

their size.

Several deposition techniques tried were spin-coating and ultrasonication. For spin coat-

ing approximately 2µl of a desalted solution are dropped onto a wafer. The wafer is then

rotated on the spinner for about 15min until the water has evaporated. The silicon wafer,

CHAPTER 8. ATOMIC FORCE MICROSCOPY

Figure 8.3: Commercial 80nm silver spheres dispersed onto a silicon waver imaged by SEM,

magnification: 30000. The spheres aggregate to large three-dimensional clusters.

that is originally hydrophobic, has to be treated in advance to become hydrophilic: Oth-

erwise the drop does not cover the whole wafer and the risk to slide off the wafer during

duration is increased. For the second technique, 2µl of a desalted solution are dropped

onto a hydrophobic silicon wafer floating on a ultrasonic bath that induces movement

of the clusters in the solution and therefore decreases the agglomeration tendency. Both

methods yielded good results for commercial 20nm silver spheres.

8.2.2

Functionalization of Silicon Wafers

A different method to overcome the challenges described above is functionalization of the

wafer with molecules with sticky ends. A drop of the solution is then brought in contact

with the functionalized surface. Due to Brownian motion the clusters randomly hit the

sticky surface and get bound. Afterward the sample is rinsed with water to remove salt

residues.

The silicon surfaces are functionalized with (3-Mercaptopropyl)trimethoxysilane (MPTMS,

linear chemical formula HS(CH

)

Si(OCH

)

, provided by Aldrich) so that the silane

groups stick to the wafer surface whereas the sulfide groups can bind the silver clusters.

The fabrication follows in principle the procedure described by [PQGO02a], but we opti-

mized the parameters for our setup: After 15min of cleaning in a piranha solution (15ml

, 35ml H

), a silicon wafer is dried by an argon flow and immediately positioned

on a grid in a 50ml polyproylene reaction tube with a 4mm hole on top. Due to an ar-

gon flow of 2l/min the evaporation rate of 20µl MPTMS, placed just below the wafer, is

enhanced. The molecules are directed upward by the Argon and a turbulent flow makes

some of the molecules settle down on the wafer surface (facing upward, i.e., away from

the MPTMS droplet) forming a self-assembled layer. This very fast and easy technique

to functionalize silicon wafers is schematically shown in fig. 8.4.

8.3. RESULTS

Figure 8.4: a) MPTMS molecules are evaporated and hit the cleaned silicon surface. Some of

the methoxy-groups dissolve from the rest of the molecule.

b) The Silicon atoms can bind to oxygen atoms in the water layer on the surface.

After 30min of functionalization, the wafers are put in an ultrasonic bath for 30min

to remove unspecifically bound MPTMS molecules and to break weak hydrogen bonds

between the MPTMS-molecules. The molecules then rearrange and form a covalently

bound network.

AFM images of the functionalized wafers are shown in Appendix C.

Solution containing metallized DNA is placed on a functionalized wafer such that it covers

the whole surface. When a silver cluster on the DNA, that undergoes diffusion, randomly

hits a sulfur atom, it gets bound to it. After some hours, the whole sample is washed with

water to remove salt. It is dried with nitrogen and then investigated by AFM. Afterward

the sample is stored in vacuum.

8.3

Results

8.3.1

Ultrasonication

Fig. 8.5 shows a silicon waver onto which UV-metallized 23mer dsDNA was dispersed.

The dispersion was done by putting a 2µl drop of metallized DNA solution onto a waver

and keeping the waver in an ultrasonic bath until the water was evaporated.

Wires with a length of several hundred nanometers and a diameter of approximately 15

to 20nm can be seen all over the AFM image 8.5. The green arrows point to two very

pronounced structures. Section analysis (not shown here) indicates a substructure of the

wires: They consist of 10 to 16nm long subunit as shown schematically in fig. 8.8. As they

CHAPTER 8. ATOMIC FORCE MICROSCOPY

Figure 8.5: Metallized 23mer dsDNA dispersed onto a silicon waver, showing parallel agglomer-

ation

do only appear in solutions containing metallized DNA but not in reference solutions, they

can be assigned to metallized DNA molecules that are agglomerated. The agglomeration

is not random but linear which might be traced back to dipole interactions among the

nanoparticles. The structures are not distributed homogeneously on the waver, they are

located to a very small area. An explanation for this is, that due to surface tension the

drop carries the DNA along when shrinking while evaporation.

8.3.2

MPTMS-Silanization

The silanization parameters were optimized to a MPTMS amount of 20µl, an Argon flow

of 2l/min and a silanization time of 30min and 30min in an ultrasonic bath, see Appendix

C. Afterward 20µl of the solution with the metallized DNA was put on the surface and

removed again several hours later. A measurement series, varying the application time,

showed that already after 2min DNA has bound to the silane layer.

Fig. 8.6 shows typical AFM images for UV-metallized 48mer dsDNA: Therefore 1µM

dsDNA was hybridized in aqueous 50mM sodium nitrate solution, that was titrated to

a pH between 7 and 8 by addition of NaOH. The silver-to-base ratio was 100:1, the

irradiation time was 10min. UV/vis spectra taken before the AFM investigation showed

a plasmon peak at 440nm.

The 5µm x 5µm scan, fig. 8.6 a), shows large clusters in the range of some hundreds

of nanometers. Next to them some lower, chain-like structures can be seen. As these

structures are hardly detectable due to the high z-range, the interesting part with many

chain-like structures is encircled with green color. These long, thin structures can be found

all over the samples, the images b) and c) show regions with high concentration. Fig. c) is

a zoom in these structures. Noise limits the maximum achieved resolution. Therefor the

composition of the long structures cannot be resolved. Thus, all AFM images show wire-

like structures with a diameter of ca. 14nm and lengths of several hundreds of nanometers.

The heights vary between 1.5 and 5nm. Some single particles are observable.

8.3. RESULTS

(a) Typical 5µm x 5µm scan

(b) Typical 1µm x 1µm scan

(d) Typical 340.5nm x 340.5nm scan

Figure 8.6: Typical AFM images of metallized 48mer dsDNA on functionalized silicon wafers.

Detailed information about the sample can be found in the text. The metallized DNA particles

agglomerate in one dimension and form long wires. Several single particles are observable.

These chain-like structures can be attributed to one-dimensional agglomerations of the

metallized DNA as shown in fig. 8.8, that can be induced by dipole-dipole-forces among

the nanoparticles. The few non-agglomerated particles have a length of 14 to 16nm and

a diameter of 7 to 9nm. As the AFM tip broadens the structures by roughly 2nm the

real dimensions are 12 to 14nm in length and 5 to 7nm in diameter. The length of the

unmetallized DNA is 16nm, its diameter is 2nm. Thus the metallized particles appear

slightly shorter. The broadening of the diameter during metallization meets the expecta-

tions. In fig. 8.6 c) structures with a length of 16nm and a width of 14nm appear. These

can be attributed to two metallized oligonucleotides, that are stacked together, see fig.

8.8b. Due to its rod-like geometry the height of the DNA structures is expected to equal

its width. This is true for the maximum heights measured as 5nm. As the MPTMS layers

are neither tight nor exactly smooth, it is possible that the DNA molecules sink in the

layer. This decreases the measured heights.

Fig. 8.7 shows an AFM image of DNA metallized in 10mM Tris buffer. As oligonucleotides

CHAPTER 8. ATOMIC FORCE MICROSCOPY

Figure 8.7: Typical AFM images of metallized 48mer dsDNA on functionalized silicon wafers.

Detailed information about the sample can be found in the text. Single particles, arranged as a

ring, can be seen, they are indicated by a green arrow. The height as well as the phase image

are shown.

again 1µM 48mer dsDNA was used, the silver-to-base-ratio was 5:1, the sample was irra-

diated for 10min.

The height as well as the phase image are shown. In phase imaging the phase of of

the cantilever oscillation during a scan is recorded. This provides information about the

composition of the material, its hardness, adhesion and many more properties and yields

images with an enlarged resolution compared to normal Tapping mode images. Details

about this powerful technique can be found in [BP04].

Whereas in the height image only one large homogeneous ring-like structure, indicated by

the green error, is visible, in the phase image single nanoparticles can be resolved: The

structure consists of about 50 nanoparticles with 10nm length and 6nm width.

The dimensions of these particles are in accordance with the values found in fig. 8.6. One

difference between the two samples is the way of agglomeration: Now the DNA sample

are agglomerated along their long axis, as can be seen in fig. 8.8c.

Fig. 8.9 shows another typical AFM image of the same sample. Now the agglomeration is

again parallel.

The section analysis shows that the elongated structures are built of several clusters. The

width of these single clusters is between 5 and 10nm. This confirms the statement, that

these chain-like structures are built of metallized DNA molecules.

8.3. RESULTS

Figure 8.8: Schematic of different possibilities for one-dimensional agglomeration of metallized

DNA:

a) shows parallel agglomeration. This seems to be the most frequent type. Next to the strands

with lengths of hundreds of nanometers, some individual particles are observable.

b) shows clusters built of 2 individual parallel particles.

c) shows agglomeration along the particle axis

Figure 8.9: In the left part a typical AFM image of UV-metallized DNA (48mer, in Tris buffer,

silver-to-base 5:1, 10min irradiation) is shown. Many typical chain-like structures can be seen. In

the right part two section analysis along one of the chainlike structures are shown: The structure

is built of several smaller units with a width of 5 to 10nm

CHAPTER 8. ATOMIC FORCE MICROSCOPY

Figure 8.10: Height and amplitude AFM images of UV-metallized DNA that was deposited on

a piranha-cleaned silicon wafer by spin-coating. Large round particles as well as small elongated

particles can be seen.

8.3.3

Spin-Coating

2µl of UV-metallized DNA were deposited onto a rotating piranha-cleaned silicon wafer.

Fig. 8.10 shows a typical AFM image.

Large round particles with a diameter of 40 to 50nm are present on the surface as well as

small particles with a length of 12 to 14nm and a width of 7nm. Particle heights of 5nm,

7nm and 14nm can be found for the small particles.

The two particle sizes are in great accordance with the CONTIN analysis done for UV-

metallized DNA as presented in 5.2.2. The dimensions of the smaller particles are the same

as found for the MPTMS-functionalized samples. The height of the particles is, however,

increased, because there is no soft molecule layer where the particles can cave in.

Thus various UV-metallized DNA samples were deposited on silicon wafers. Three differ-

ent techniques - the ultrasonic bath method, spin coating and the use of functionalized

surfaces - were applied, whereby the last one achieved best reproducible results. Besides

some large spherical particle in the range of some tens to some hundred of nanometers,

small elongated particles can be found in solution. The larger particles are assigned to

unspecific silver depositions and agglomerations, whereas the smaller particles are met-

allized DNA molecules: They are 10 to 16nm long and 5 to 7nm broad. The measured

heights differ depending on the method of deposition. Assuming that the nanoparticles

can sink in soft molecular layers, all heights are in the range between 5 and 14nm. The

metallized DNA molecules tend to aggregate by building chain-like structures.

Chapter 9

Conclusion

Metallized DNA structures were produced by two different methods, photoinduced silver

deposition and aldehyde-modified Tollens metallization. Afterward they were character-

ized by optical techniques as well as atomic force microscopy.

All metallized DNA samples show characteristic plasmonic peaks between 405 and 440nm

in the absorption spectrum indicating silver particles in the nanometer range. These peaks

only occur in samples containing DNA (for the unspecific UV -metallization) or modified

DNA (for the Tollens-metallization) confirming that the nanoclusters are grown on the

template and not randomly in solution.

Conditions for a reproducible and efficient synthesis of nanoparticles have been found: The

use of a buffer solution provides reproducibility; and an alkaline environment enhances

the silver reduction rate.

Comparison of the peak positions and bandwidths of UV-metallized samples with calcu-

lated values from Mie theory indicate that the grown silver nanoparticles have radii of

only a few nanometers. Moreover, the spectra show that dsDNA can be metallized more

efficiently than ssDNA and yields slightly larger particles. The resulting particles, i.e. their

size distribution and intensity, also depend on the specific base sequence as experiments

with several samples with varying GC-content show. Therefore not only the fraction of

GC-bases influences the nanoparticle growth but also the neighboring bases. By decon-

voluting the absorption spectra to eliminate overlapping effects caused by the DNA and

nitrate absorption, two peaks can be determined. The low-intensity peak at short wave-

lengths is attributed to transverse plasmons, the high-intensity peak at long wavelengths is

attributed to longitudinal plasmons. This indicates an either rod-like or chain-like shape of

the metallized DNA molecules: This is in accordance with electrostatic calculations except

that the measured peaks occur at much shorter wavelengths than expected. Deconvoluted

absorption spectra show that the axis ratio of the particles can be in principle tuned

by varying one of the following parameters: length of the used DNA, amount of added

silver or irradiation time. An applicable theory allowing for quantitative determination

of the axis ratios is missing. Polarized Dynamic Light Scattering measurements quali-

tatively confirm an asymmetric particle shape. Unpolarized measurements show that at

CHAPTER 9. CONCLUSION

least three different particle sizes - hydrodynamic radii around 1nm, 8nm and 30nm - are

present in solution, whereby the smaller particles are more frequent. With increased DNA

length or extended irradiation time the particles become larger. The addition of more

silver produces slightly larger particles with a much narrower size-distribution indicating

that a stable cluster size has been reached.

The reaction rate of the Tollens-metallization process crucially depends on the amount of

ammonium present in the reagent: By increasing this amount, the reaction is slowed and

the plasmonic peak intensity, i.e. the number of particles or their size, is reduced. More-

over, the reaction speed depends on the silver concentration as well as the density of the

modified bases. The reaction can be accelerated and its maximum absorbance enhanced

by adding a development solution. Absorption spectra as well as DLS measurements in-

dicate that the solution is not stable and an aggregation of the individual clusters sets in

after some hours. Proof-of-principle-experiments with sodium citrate showed that it can

be possibly applied as a developer as well as a stabilizing agent. Absorption spectra of

Tollens-metallized DNA samples show a plasmonic peak that is broadened and red-shifted

compared to the spectra taken after UV-metallization. DLS spectra indicate a broadened

particle distribution compared to the UV/vis spectra. Despite from large clusters in the

micrometer range, which can be attributed to dirt in the solution, only one particle size

is present in the solution. Monitoring the growth process temporally with DLS, one can

see that first clusters build within few minutes. The average particle size decreases with

time whereas the intensity increases. Contrary to UV-metallized DNA, no second peak

appears after deconvolution. The reason might be that the plasmonic peak width is too

broad to resolve another peak nearby. DLS studies, however, confirm the rod- or chainlike

character.

Structural characterization was done by Atomic Force Microscopy: Particles with a length

of 14nm and a diameter of 5nm were found and attributed to the metallized DNA. The

length is shorter than the nominal DNA length of 16nm, the diameter is broadened from

2nm to about 5nm. Most particles were aggregated parallel to one-dimensional structures

with lengths of several hundreds of nanometers

Chapter 10

Outlook

Metallization Environment

The requirements for the solution in the UV-metallization are the ability to keep the

pH constant, a slightly basic pH value and a preferably low salt concentration. Tris

buffer/NaCl matches most of these claims, but nevertheless the amount of added silver is

limited by precipitation of silver chloride. Thus a buffer that fulfills the requirements and

that does not contain any compounds building precipitations with silver has to be found.

Stabilization

First experiments showed that stabilization with sodium citrate works in principle. In

further experiments the optimum parameters for the stabilization have to be found out.

Development Solution

Adding a development solution to Tollens-metallized samples turned out to be a promising

method for enhancing the absorption peaks. The outcome are blue-shifted, comparatively

narrow peaks that stand for small particles. DLS measurements with Development Solu-

tions are still pending. Moreover, different acids to stop the reaction in a controlled way

have to be tested. Sodium citrate might be used as a stabilizer as well as a developing

agent at the same time.

Separating the Particles

Recent DLS measurements confirmed the existence of up to three different particle sizes

in solution. The largest ones, that have radii of typically 30 to 50nm, are also present

in reference samples, whereas the smaller ones can be attributed to nanoclusters grown

on DNA. Although the small clusters are much more frequent, the effective signal is very

sensitive to the larger particles, as the absorption intensity is proportional to r

and the

scattering intensity even to r

. Therefore the particles shall be separated by HPLC and

afterward being analyzed again. The DLS correlation times in both polarization modes

can then be compared to the calculated values presented in chapter 5.3.1.

Structural Studies

AFM images gave first information about metallized DNA. Even better resolution can,

however, be achieved with High Resolution Transmission Electron Microscopy (HRTEM).

CHAPTER 10. OUTLOOK

The challenge to overcome in this method is the homogeneous distribution of the particles

on the TEM-grid.

More Complex Structures

Linearized DNA has extensively been studied in this thesis. Also, first experiments with

circular DNA have been made and analyzed by DLS as well as absorption spectrometry

showing commonalities as well as differences. In future, more complex DNA structures

shall be metallized.

Appendix A

DNA Sequences

Used DNA sequences:

23mer:

GCC GGT CCT GTT ACT TGT GGC GC

38mer: GCC GGT CCT GTT ACT TGT GGT CCT GTT ACT TGT GGC GC

48mer, seq1: TAG TCG TAA GCT GAT ATG GCT GAT TAG TCG GAA GCA TCG AAC

GCT GAT

48mer, seq2: TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT

TTT TTT

48mer, seq3: GGG TGG GTG GGT GGG TGG GTG GGT GGG TGG GTG GGT GGG TGG

GTG GGT

96mer: TAG TCG GAA GCA TCG AAC GCT GAT TAG TCG TGA GCA CAT GGA CCT

GAT TAG TCG TAA GCT GAT ATG GCT GAT TAG TCG GAA GCA TCG AAC GCT

GAT

50mer MB: GGC CGC CGC CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TGG

CGG CGG CC

80mer MB: GGC CGC CGC CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT

TTT TTT TTT TTT TTT TTT TTT TTT TTT TGG CGG CGG CC

Appendix B

Reference Samples

B.1

Absorption Spectroscopy

Figure B.1: Absporption spectrum of 10mM Trisbuffer/HCl with 1mM AgNO

after 45min of

irradiation. The absorption is in the same range as the background of the samples with metallized

DNA. The spectrum is an overlap of two features: Around 300nm nitrate absorbs. Above ca.

300nm the absorption is raised due to unspecific silver deposition. In contrast to the metallized

DNA samples there is no plasmonic peak between 400 and 420nm.

B.1. ABSORPTION SPECTROSCOPY

Figure B.2: UV/vis spectra of 10mM Tris buffer solutions containing 1mM silver nitrate and

sodium citrate/PVP. The stabilizer was added before the 10min of irradiation. The spectra show

plasmonic peaks at 414/425nm.

Figure B.3: UV/vis spectra of 10mM Tris buffer solutions containing 1mM silver nitrate and

sodium citrate. The stabilizer was added directly after the 10min of irradiation. The spectra

show no plasmonic peak. The peak seen around 300nm can be attributed to the absorption of

nitrate.

APPENDIX B. REFERENCE SAMPLES

Figure B.4: UV/vis spectra of unmodified 38bp DNA in aqueous solution to which an aliquot

of a Tollens reagent containing 3.2mM Ag is added. Within 45min no peak occurs. Adding a

Development solution slightly increases the absorption.

B.2

DLS

[H]

Figure B.5: Commercial 20nm silver spheres in a concentration of 1.2nM were analyzed with

the ALV DLS setup. The graph shows a CONTIN calculation of the number weighted radius.

According to the manufacturer the deviations from the mean diameter are ±5nm.

B.3. AFM

B.3

AFM

(a) image1

(b) image1

Figure B.6: 5µm x 5µm AFM micrographs of a reference sample on DNA, z-range: 10nm

100µM of silver nitrate was added to standard buffer. Afterward 20µl of this solution were put

on a silanized silicon wafer and let react there for 30min. The sample was rinsed with pure

water, dried with nitrogen gas and analyzed by AFM. The micrograph shows some unspecific

silver deposition and maybe salt residues but no elongated structures as on the metallized DNA

samples.

Appendix C

Parameters of MPTMS-Silanization

Fig. C.1 shows a silicon wafer after 15min Piranha (35ml H

, 15ml H

) cleaning.

Figure C.1: Piranha-cleaned silicon wafer, 1µm × 1µm scan, z-range 3nm

The MPTMS amount was varied between 10µl and 100µl. The higher the amount of MPTMS,

the more heterogeneous the surface of the sample is: The molecules form large aggregates on the

substrate. Thus the amount of MPTMS was set to 20µl.

As already mentioned by Pavlovic [PQGO02b] the number of molecules on the wafer surface is

independent of the silanization time. For the optimization of the parameters, the silanization

time was varied between 10min and 60min. For the experiments a medium time of 30min was

chosen. The argon flow was set to 2l/min.

Typical images recorded after silanization are shown in fig. C.2: Large cluster, that appear

on the relatively flat surface, can be identified as excess MPTMS molecules that have formed

aggregates. Holes with diameters of some tens of nanometers up to a few micrometers confirm

that self-assembled layers have been deposited on the wafer.

Measuring the depths of the holes in comparison to the upper layer one finds steps of roughly

2.0nm, 2.5nm, 3.5nm and 5.2nm. The theoretical length of the MPTMS-molecule is 0.77nm.

Granted that 5.2nm is the maximum layer thickness on the analyzed surfaces, one finds layers

of two (step = 2.0nm), four (step = 3.5nm) and six (step = 5.2nm). The holes might originate

(a) after silanization, 1µm ×

1µm scan, z-range 10nm

(b) after silanization, 5µm ×

5µm scan, z-range 10nm

Figure C.2: Surfaces directly after silanization. Functionalization parameters are20µl MPTMS,

2l/min Argon for 60min

from holes in water layer. Assuming that the water layer is about 0.4nm, the theoretical and

experimental values match quite well.

After silanization the wafers were put into the ultrasonic bath for 30min. Thereby the large

aggregates were removed from the surface. An image of a wafer after the ultrasonic bath can be

seen in fig. C.3.

Figure C.3: Silanized silicon wafer after 30min ultrasonication. Functionalization parameters

are 20µl MPTMS, 2l/min Argon flow for 30min. The image represents a 5µm × 5µm scan, the

z-range is 3nm.

Appendix D

Acknowledgments

An diese Stelle m¨

ochte ich noch all jenen danken, die mich w¨

ahrend meiner Diplomarbeit un-

terst¨

utzt haben und zum Gelingen dieser Arbeit beigetragen haben.

Prof. Dr. Gerhard Abstreiter danke ich f¨

ur die M¨

oglichkeit, an diesem großartigen Institut

arbeiten und meine Diplomarbeit an seinem Lehrstuhl anfertigen zu k¨

onnen.

Dr. Ulrich Rant gilt mein besonderer Dank: f¨

ur die ¨

außerst interessante Themenstellung, die

gute Arbeitsatmosph¨

are sowie den großen Freiraum, den ich bereits w¨

ahrend meiner Diplomar-

beit genießen durfte.

Den Verantwortlichen vom Lehrstuhl E25 danke ich daf¨

ur, dass ich sowohl ihr AFM als auch das

UV/vis Absorptionsspektrometer f¨

ur teilweise sehr lange Zeit belegen durfte. Dr. Paul Berberich

vom Lehrstuhl E10 danke ich f¨

ur die Bereitstellung des REM sowie f¨

ur die große Hilfsbere-

itschaft, die ich bei allen Fragen und Problemen erfahren durfte. Dr. Nikolaus Neumaier,

vom Chemie-Department der TU M¨

unchen, danke ich f¨

ur die Einf¨

uhrung am DLS-Ger¨

at. Dr.

Marie-Sousai Appavou und Dr. Wolfgang Doster vom Lehrstuhl E13 gilt mein besonderer

Dank daf¨

ur, dass ich kurzfristig ihren DLS-Aufbau verwenden konnte und f¨

ur die vielen netten

Diskussionen.

Martina Hofmann f¨

ur die große Hilfe mit der Silanisierung und den AFM-Aufnahmen sowie

die netten Gespr¨

ache zwischendurch.

Anna und Erika f¨

ur ihre geduldige Hilfe bei all meinen chemischen Fragen. Sebastian, Daniel,

Rocio, Jelena, Wolfgang und Kenji f¨

ur die tolle Arbeitsatmosph¨

are.

Ade Ziegltrum f¨

ur den Bau der Silanisierungsanlage sowie die Hilfe bei vielen Kleinigkeiten.

Irmgard f¨

ur ihre stetige Hilfsbereitschaft und deren Arbeit im Hintergrund viel zur guten

Arbeitsatmosph¨

are beitr¨

agt.

Christian Wirges vom Chemie-Department der LMU danke ich f¨

ur die vielen fruchtbaren

Diskussionen ¨

uber unsere Arbeit.

Konrad f¨

ur die vielen Gespr¨

ache fachlicher und nichtfachlicher Art.

Zu guter Letzt m¨

ochte ich meiner Familie sowie Friedrich f¨

ur all ihre Geduld und Un-

terst¨

utzung herzlich danken.

100

Bibliography

[AN05]

N.W. Ashcroft and Mermin N.D. Festk¨

orperphysik. Oldenbourg, 2005.

[BAF05]

L. Berti, A. Alessandrini, and P. Facci.

Dna-templated photoinduced silver

deposition. Journal of the American Chemical Society, 127(32):1121611217,

2005.

[BESBY98]

E. Braun, Y. Eichen, U. Sivan, and G. Ben-Yoseph. Dna-templated assembly

and electrode attachment of a conducting silver wire. Nature, 391(6669):775778,

1998. 30.

[BGM

06]

G.A. Burley, J. Gierlich, M.R. Mofid, H. Nir, S. Tal, Y. Eichen, and T. Carell.

Directed dna metallization.

Journal of the American Chemical Society,

128(5):13981399, 2006.

[BH83]

C.F. Bohren and D.R. Huffman. Absorption and Scattering of Light by Small

Particles. WILEY-VCH, 1983.

[BKRS06]

J. Boleininger, A. Kurz, V. Reuss, and C. Sonnichsen. Microfluidic continuous

flow synthesis of rod-shaped gold and silver nanocrystals. Physical Chemistry

Chemical Physics, 8(33):38243827, 2006. Boleininger, Johann Kurz, Andreas

Reuss, Valerie Soennichsen, Carsten 42.

[Blu03]

R. Blume.

Prof. blumes bildungsserver f¨

ur chemie.

http://dc2.uni-

bielefeld.de/dc2/tip/12 00.htm, 2003. [Online; accessed 14-June-2007].

[BP04]

K.I. Babcock and C.B. Prater. Phase imaging: Beyond topography. Veeco Ap-

plication Note, 2004. [AN11, RevA1].

[ES68]

Gunther L. Eichhorn and Yong Ae Shin. Interaction of metal ions with polynu-

cleotides and related compounds. xii. the relative effect of various metal ions on

dna helicity. Journal of the American Chemical Society, 90(26):73237328, 1968.

[Fli00]

T. Fliessbach. chapter 6. Spektrum, third edition, 2000.

[GBP89]

J. P. Goudonnet, J. L. Bijeon, and M. Pauty. A method for determining shape

and volume of ellipsoidal silver particles from absorbance measurements. Thin

Solid Films, 177:4957, 1989. 13.

[HBSG64]

R. Huisgen, H. O. Bayer, F. C. Schaefer, and H. Gotthardt.

New type of

mesoionic aromatic compound + its 1,3-dipolar cycloaddition reactions with

acetylene derivatives.

Angewandte Chemie-International Edition, 3(2):136,

1964. 10.

101

BIBLIOGRAPHY

[HG99]

A. Henglein and M. Giersig. Formation of colloidal silver nanoparticles: Capping

action of citrate. Journal of Physical Chemistry B, 103(44):95339539, 1999. 16.

[JC72]

P. B. Johnson and R. W. Christy. Optical constants of the noble metals. Phys.

Rev. B, 6(12):43704379, Dec 1972.

[KB04]

K. Keren and E. Braun. Sequence-specific molecular lithography towards dna-

templated electronics.

Chemical Engineering and Technology, 27(4):447452,

2004. 12.

[KV83]

U. Kreibig and M. Vollmer. Optical Properties of Metal Clusters. Springer, 1983.

[LES99]

S. Link and M. A. El-Sayed. Spectral properties and relaxation dynamics of

surface plasmon electronic oscillations in gold and silver nanodots and nanorods.

Journal of Physical Chemistry B, 103(40):84108426, 1999. 133.

[Lin06]

S. Lingitz. Electronic transport investigations on conjugated molecules based on

nanogap electrode devices, 2006.

[LKSH00]

T. Liebermann, W. Knoll, P. Sluka, and R. Herrmann.

Complement hy-

bridization from solution to surface-attached probe-oligonucleotides observed by

surface-plasmon-field-eahanced fluorescence spectroscopy. Colloids and Surfaces

a-Physicochemical and Engineering Aspects, 169(1-3):337350, 2000. 37.

[Man78]

G. S. Manning. Molecular theory of polyelectrolyte solutions with applications

to electrostatic properties of polynucleotides. Quarterly Reviews of Biophysics,

11(2):179246, 1978. 92.

[MBS

02]

J. J. Mock, M. Barbic, D. R. Smith, D. A. Schultz, and S. Schultz. Shape

effects in plasmon resonance of individual colloidal silver nanoparticles. Journal

of Chemical Physics, 116(15):67556759, 2002. 18.

[MKT03]

S.A.E Marras, F.R. Kramer, and s. Tyagi. Genotyping single nucleotide poly-

morphisms with molecular beacons, volume 212. The Humana Press Inc., 2003.

[MP04]

M. Mertig and W. Pompe.

Biomimetic Fabrication of DNA-based Metallic

Nanowires and Networks, chapter 17. WILEY-VCH, first edition, 2004.

[MPJG

06]

P. Mulvaney, J. P´

erez-Juste, M. Giersig, L.M. Liz-Marz´

an, and Pecharrom´

an C.

Drastic surface plasmon mode shifts in gold nanorods due to electron charging.

Plasmonics, 1(1):15571955, March 2006.

[MVH99]

J. B. Mills, E. Vacano, and P. J. Hagerman. Flexibility of single-stranded dna:

Use of gapped duplex helices to determine the persistence lengths of poly(dt)

and poly(da). Journal of Molecular Biology, 285(1):245257, 1999. 62.

[Noy05]

M. Noyong. Synthese und Organisation von Gold-Nanopartikeln mittels DNA.

PhD thesis, TU Dreseden, 2005.

[PQGO02a]

E. Pavlovic, A. P. Quist, U. Gelius, and S. Oscarsson. Surface functionalization of

silicon oxide at room temperature and atmospheric pressure. Journal of Colloid

and Interface Science, 254(1):200203, 2002. 18.

102

BIBLIOGRAPHY

[PQGO02b]

E. Pavlovic, A. P. Quist, U. Gelius, and S. Oscarsson. Surface functionalization of

silicon oxide at room temperature and atmospheric pressure. Journal of Colloid

and Interface Science, 254(1):200203, 2002. 18.

[Pro82]

S. W. Provencher. Contin - a general-purpose constrained regularization program

for inverting noisy linear algebraic and integral-equations. Computer Physics

Communications, 27(3):229242, 1982. 6.

[PWLW02]

F. Patolsky, Y. Weizmann, O. Lioubashevski, and I. Willner. Au-nanoparticle

nanowires based on dna and polylysine templates.

Angewandte Chemie-

International Edition, 41(13):23232327, 2002. 29.

[QD05]

L. T. Qu and L. M. Dai. Novel silver nanostructures from silver mirror reaction

on reactive substrates. Journal of Physical Chemistry B, 109(29):1398513990,

2005. 18.

[RABdA06]

I. Romero, J. Aizpurua, G. W. Bryant, and F. J. G. de Abajo. Plasmons in

nearly touching metallic nanoparticles: singular response in the limit of touch-

ing dimers. Optics Express, 14(21):99889999, 2006. Romero, Isabel Aizpurua,

Javier Bryant, Garnett W. Garcia de Abajo, F. Javier 44.

[Ran05]

U. Rant. Electrical manipulation of dna-layers on gold surfaces. PhD thesis, TU

Munich, 2005.

[Ric03]

J. Richter.

Metallization of dna.

Physica E-Low-Dimensional Systems and

Nanostructures, 16(2):157173, 2003. 73.

[SCB96]

S. B. Smith, Y. J. Cui, and C. Bustamante. Overstretching b-dna: The elas-

tic response of individual double-stranded and single-stranded dna molecules.

Science, 271(5250):795799, 1996. 29.

[SGHURS

05] A. Slistan-Grijalva, R. Herrera-Urbina, J. F. Rivas-Silva, M. Avalos-Borja, F. F.

Castillon-Barraza, and A. Posada-Amarillas. Classical theoretical characteriza-

tion of the surface plasmon absorption band for silver spherical nanoparticles

suspended in water and ethylene glycol. Physica E-Low-Dimensional Systems

and Nanostructures, 27(1-2):104112, 2005. 38.

[TG80]

M. M. Tirado and J. Garciadelatorre. Rotational-dynamics of rigid, symmet-

ric top macromolecules - application to circular-cylinders. Journal of Chemical

Physics, 73(4):19861993, 1980. 27.

[Tin60]

Ignacio Tinoco. Hypochromism in polynucleotides. Journal of the American

Chemical Society, 82(18):47854790, 1960.

[TK96]

S. Tyagi and F. R. Kramer. Molecular beacons: Probes that fluoresce upon

hybridization. Nature Biotechnology, 14(3):303308, 1996. 23.

[vdZDBP00]

B.M.I. van der Zande, J.K.G. Dhont, M.R. Bohmer, and A.P. Philipse. Col-

loidal dispersions of gold rods characterized by dynamic light scattering and

electrophoresis. Langmuir, 16(2):459464, 2000.

103

BIBLIOGRAPHY

[Wik06]

Wikipedia.

Dna

wikipedia,

the

free

encyclopedia.

http://en.wikipedia.org/w/index.php?title=DNAoldid=138027697,

2006.

[Online; accessed 26-October-2006].

[Wik07]

Wikipedia.

Persistence

length

wikipedia,

the

free

encyclopedia.

http://en.wikipedia.org/w/index.php?title=Persistencelen gth&oldid=134452654,

2007. [Online; accessed 15-June-2007].

[Zuk03]

M. Zuker. Mfold web server for nucleic acid folding and hybridization prediction.

Nucleic Acids Research, 31(13):34063415, 2003. 42.

104

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Details

Seiten
Erscheinungsform: Originalausgabe
Jahr: 2007
ISBN (eBook): 9783836622127
DOI: 10.3239/9783836622127
Dateigröße: 2.8 MB
Sprache: Englisch
Institution / Hochschule: Technische Universität München – Physik Department, Walter Schottky Institut
Erscheinungsdatum: 2008 (November)
Note: 1,0
Schlagworte: optical properties dynamic light scattering silver nanoparticles uv/vis

Autor

Nadine Kammerlander (Autor:in)

Optical Properties of metallized DNA

Zusammenfassung

Leseprobe

Inhaltsverzeichnis

Details

Autor

Nadine Kammerlander (Autor:in)