Assembly and Characterization of Protein Multi-layer Systems

Danica Christensen

Masters Thesis

September 14, 2004

Chemistry of Materials Masters Program Johannes Gutenberg University-Mainz

Max Plank Institute for Polymer Research

1 Abstract This work was completed as part of the requirements for a Masters of Science degree from the

program entitled Chemistry of Materials at the Johannes Guttenberg University in Mainz,

German.

The aim of the project was to build multi-layer protein architectures with streptavidin (SA)

and biotinylated immunoglobulin G (B-IgG). The multi-layers were characterized using

surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS).

Neutron reflectivity was also used for one experiment.

The experiments showed that the multi-layer system had a linear growth of thickness per layer

deposited. The thickness of the layers obtained was reproduced multiple times.

This thesis will also discuss the results from SPFS experiments on proteins labeled with a

chromophore. The preliminary results from an alternating polymer and protein system will

also be discussed and recommendations for further studies on these systems will be proposed.

ii

Table of Contents

1 Abstract ................................................................................................................................... ii

Table of Contents ..................................................................................................................iii

List of Abbreviations............................................................................................................. iv

2 Introduction ............................................................................................................................. 1

3 Theoretical Introduction.......................................................................................................... 3

3.1 Surface Plasmon Resonance.......................................................................................... 3 3.1.1 Total Internal Reflection ........................................................................................... 4 3.1.2 Excitation of Surface Plasmons................................................................................. 5 3.1.3 Requirements for Surface Plasmon excitation .......................................................... 7

3.2 Surface Plasmon Fluorescence Spectroscopy .............................................................. 9 3.2.1 Fluorescence............................................................................................................ 11 3.2.2 Experimental Set-Up ............................................................................................... 12

3.3 Neutron Reflectivity ..................................................................................................... 14

3.3.1 Data Analysis .......................................................................................................... 17 3.4 Materials and Methods ................................................................................................ 18

3.4.1 Streptavidin and Avidin .......................................................................................... 18 3.4.2 Immunoglobulin G .................................................................................................. 19 3.4.3 Surface modification by NHS/EDC binding........................................................... 20 3.4.4 Surface modification by biotinylated thiol binding................................................. 22

3.5 Applications .................................................................................................................. 23 4 Procedure............................................................................................................................... 24

5 Results and Discussion.......................................................................................................... 26

5.1 Thickness....................................................................................................................... 26

5.2 Structure model from Neutron reflectivity................................................................ 32

4.3 Streptavidin vs. Avidin ................................................................................................ 35

5.4 Experimental reproducibility...................................................................................... 38

5.5 Multi-layer architecture’s stability against free biotin............................................. 40

5.6 Streptavidin immobilization techniques .................................................................... 42

5.7 Thickness effects of Alexa Fluoro ............................................................................... 45

5.8 Fluorescence Effects of Alexa Fluoro ......................................................................... 46

5.9 Polymer and Protein Multi-layers .............................................................................. 53 7 Conclusion............................................................................................................................. 57

Bibliography............................................................................................................................. 59

Acknowledgments .................................................................................................................... 61

iii

List of Abbreviations

AFSA Alexa fluoro labeled streptavidin

A.U. Arbitrary units

Av Avidin

B-IgG Biotinylated immunoglobulin G

B-PEI Biotinylated polyethylenimine

B-thiol 11-mercapto-(8-biotinamido-4, 7, dioxaoctyl-)-undecanoylamide (top).

c Speed of light in vacuum

D Daltons

Dr

Dielectric displacement

DNA Deoxyribonucleic acid

θc Critical angle

E Energy

Er

Electric field vector

ε Dielectric function

EDC 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride

fwhh Full width at half height

GMO Genetically modified organism

h Plank’s constant

Hr

Magnetic field vector

hCG Human chorionic gonadotrophin

HEPES n-[2-Hydroxyethyl]piperazine-n‘-[2-ethanesulforic acid]

IC Intersystem crossing

I(q) Scattering Intensity

kr

Outgoing wave vector

0kr

Incoming wave vector

kz electromagnetic wave decay along the z-axis

λ wavelength

LANSCE Los Alamos Neutron Science Center

n refractive index

nm nanometer

NHS N-Hydroxysuccinimide

iv

φ Phase shift

PDMS Polydimethylsiloxane

PEI Polyethylenimine

qr Scattering vector

S Electronic level

σ Surface charge density

SA Streptavidin

SLD Scattering Length Density

SP Surface Plasmon

Spacer 1-mercapto-undecanole

SPEAR Surface Profile Analysis Reflectometer

SPFS Surface Plasmon Fluorescence Spectroscopy

SPR Surface Plasmon Resonance

TIR Total Internal Reflection

TOF Time of Flight

υ Frequency

VR Vibrational relaxation

v

2 Introduction

Biosensors, both immunosensors and DNA detection devices, are extensively studied and are

becoming very important detection devices for today’s society. Scientists are always trying to

perfect the ideal system for a detection scheme. A high sensitivity is the parameter most

desired, but the detection techniques should also be reusable for economic reasons. Ideally,

the sensors should also be cheap and easy to manufacture.

One important example of an immunosensor that is commonly used is the home pregnancy

test. The hormone human chorionic gonadotrophin (hCG) is detected by an antibody attached

to the surface. [1] The ELIZA test is another commonly used immunosensor used to test for

the AIDS virus. The antibodies produced by the body are probed to see if the virus is present

in the body. [2]

Genetic testing is also gaining in popularity. A recent newspaper article discussed the

implications that DNA testing on infants can have for parents. [3] The article mentions that

there are currently 30 tests that can be preformed on infants within a couple of days of birth

for the detection of genetic diseases. As more genetically engineered products are being

introduced to the market, the interest of the global community in detection schemes for

genetically modified organisms (GMOs) is increasing.

The system used for these experiments was an alternating streptavidin (SA) monolayer on a

solid surface alternated with layers of biotinylated immunoglobulin G (B-IgG). Figure 1

shows a schematic of the architecture that was used. By implementing two methods for

increasing the sensitivity, this system can have many advantages over other detection

schemes. The first method for increasing the sensitivity being the placement of the dye

molecule at an ideal distance from the metal surface to use the excited surface plasmon (SP)

to increase the emission of the chromophore and the second method being the increase of

probe density on the surface.

1

Figure 1: A schematic of the multi-layer architecture used for these experiments.

These multi-layer systems were investigated using a variety of detection techniques: surface

plasmon resonance spectroscopy (SPR), surface plasmon fluorescence spectroscopy (SPFS),

and neutron scattering. This thesis will give a short theoretical introduction into the

aforementioned techniques. The results will be discussed and explanations will be given. A

brief overview of some future work will also be included.

2

3 Theoretical Introduction 3.1 Surface Plasmon Resonance Surface plasmon resonance was used extensively during this project to measure the thickness

of the thin films deposited on the gold surface and to investigate the binding kinetics of the

layers as they were deposited. SPR is an optical technique that is highly sensitive to changes

in the dielectric material deposited onto a metal layer.

Figure 2: The Kretschmann configuration for exciting surface plasmons on a metal surface.

A monochromatic light source, in this case a He-Ne laser with a wavelength of λ=632.8 nm,

was coupled to a thin metal layer with a high refractive index prism in what is known as the

Kretschmann configuration. [4] There are other configurations that can be used to excite SPs,

but as the Kretschmann configuration was exclusively used for these experiments it will be

the only method discussed. The reflected light was collected by a photodiode detector at the

opposite side of the prism. The light source excites a surface plasmon (SP) at the interface of

the metal and the dielectric medium. A SP is an electromagnetic wave that propagates along

the interface between a metal and a dielectric medium; the distribution of the electromagnetic

field is that of an evanescent wave, figure 2. Specifically, this means the electric field

intensity of the wave decays exponentially perpendicular to the interface and is a damped

oscillatory wave along the interface, figure 3. [5]

3

Figure 3: The electric field intensity of a surface plasmon excited at the interface between a metal and a dielectric interface. [6]

The reflected light that is collected by the photodiode is plotted against the incident angle, θ,

to obtain a SP scan. A SP scan has two points of interest to note, the first is the angle of total

internal reflection or the critical angle, θc, and the surface plasmon resonance angle. See

figure 5 (a) for an example of a SP scan.

3.1.1 Total Internal Reflection The total internal reflectance (TIR) occurs because of the differences in the refractive indices

of the two materials that share an interface. TIR occurs above a critical angle, θc, when an

electromagnetic wave coming through the material with the higher refractive index (i.e. the

prism in this case) is reflected without loss from transmission into the material with the lower

refractive index material. The critical angle can be determined using Snell’s law, equation 1,

where nd is the refractive index of, for example, an aqueous medium and np is the refractive

index of the prism. The refractive indices can be found by taking the square root of the

dielectric functions, ε. Because the critical angle does not depend on the thin dielectric layer,

but only on the refractive indices of the prism and the aqueous solution, it will not change as

material is added to the surface as long as the aqueous solution is always the same. [6]

p

dc n

n=θsin (1)

iin ε= (2)

4

3.1.2 Excitation of Surface Plasmons Ideally, the intensity of the beam that is reflected at angles larger than the critical angle to the

detector is 100%, up to the angle at which the surface plasmon is excited. At the surface

plasmon resonance angle, the energy from the laser light is used to excite the SP. This

transfer of energy can be seen as a dip in the angular scan of the reflectance. The angle of

incidence that the SP is excited at is dependent on the optical properties of the dielectric

material. The angle shift will provide information on the refractive index of the material or

the thickness of the dielectric layer. The resonance angular shift is proportional to the product

of the change in thickness, ∆d, and the change in refractive index, ∆n, equation 3. With

traditional surface plasmon reflectometry, the refractive index and the thickness cannot be

probed independently with SPR, but if the refractive index is known from a separate detection

technique the thickness can be determined or vice versa. [7] It is possible with specialized

SPR methods to independently measure the distance from the refractive index. [8] It has been

shown that this can be done by using two lasers with different wavelengths.

nd ∆⋅∆∝∆θ (3)

If the change in refractive index with concentration is known from literature or refractivity

experiments, the change in mass upon adsorption of dielectric material to a surface can be

calculated using equation 4.

dcdn

nndm bd −=∆ (4)

Where nd is the refractive index of the dielectric medium, and nb is the refractive index of the

buffer. The units of dn/dc are given in ml/g.

The binding kinetics can be investigated by keeping the incident angle constant and observing

the change in the reflectance as a function of time, as the surface plasmon angle shifts. This

can be seen in figure 4 (a) and (b).

5

(a)

50 55 600

20

40

60

80

1θ2

θcθ

Ref

lect

ance

[%]

Angle [degree]

0 10 20 30 4024

26

28

30

32

34

Time [min]

Ref

lect

ance

[%]

0

2

4

Thickness [nm]

(b)

Figure 4 (a) shows two SPR curves, one taken before deposition of thin film, θ1, and the other taken afterwards, θ2. (b) The kinetics of the deposition can be investigated by observing the change in reflectance as the SPR shifts while keeping the angle constant.

6

3.1.3 Requirements for Surface Plasmon excitation The TIR that occurs because of the difference in the high refractive index of the prism and the

lower refractive index of the aqueous medium is essential for the excitation of a surface

plasmon. The TIR allows the energy from the incident light to be transferred to the interface

between the prism and the metal layer in the form of an evanescent wave. The existence of an

evanescent wave is governed by the dispersion relation; this relationship between the angular

frequency and the propagation constant is shown in figure 5. The evanescent wave can excite

the SP if the dispersion relationship at the boundary between the metal and the dielectric

material is met.

Figure 5: The dispersion relation that determines the excitation of an evanescent wave.[7]

One restriction to the excitation of surface plasmons is described by equation 5 and states that

the dielectric displacement, Dr

, of the electromagnetic mode must have a component

perpendicular to the surface, where σ is the surface charge density. This indicates that only a

p-polarized light source will excite a SP; the electric field component in p-polarized light

normal to the surface, with the magnetic field vector being parallel to the surface.

( ) πσ412 =⋅− zDD rrr

(5)

The surface electromagnetic wave is given by equations 6 and 7, where the vector A

incorporates both the electric field vector, Er

, and the magnetic field vector, Hr

. 1Ar

is the

equation for the electromagnetic wave in the first material and 2Ar

is for the second material.

7

The wave vectors in the x-direction are given by 1xkr

and k 2x

r, and likewise the k 1z

r and 2zk

r

vectors are the wave vectors in the z-direction. ω is the angular frequency.

0

0>

,)(

10111 <= −+ zeAA tzkxki zx ωrrrrrr

(6) ,)(

20222= −+ zeAA tzkxki zx ωrrrrrr

(7)

The surface wave also must follow the Maxwell equations; both the electric and magnetic

field vectors.

0=⋅∇ H

r (8)

0=⋅∇ E

r (9)

01=

∂∂

+×∇t

Hc

Er

r (10)

0=∂∂

−×∇tE

cH

rr ε (11)

Because the electric field and the magnetic field components must be equal at the interface,

the wave vectors in the x-direction must also be equal (i.e. kx1=kx2=kx). From equations 6, 7,

and 10 we can get equations 12 and 13.

1111 xzz Ec

Hk εω= (12)

2222 xzz Ec

Hk εω−= (13)

2

1

2

1

εε

−=z

z

kk (14)

Equation 14 is determined from equations 12 and 13 and states that the dielectric constants of

the materials must have opposite signs to excite a surface plasmon. The dielectric constants

are composed of a real, εi’, and an imaginary part, εi”. For the interface between a metal and a

dielectric material the rule described by equation 14 holds true; the dielectric constant for a

metal is a negative number and for a dielectric material it is positive. [6]

8

3.2 Surface Plasmon Fluorescence Spectroscopy Surface Plasmon Fluorescence Spectroscopy utilizes the evanescent field from the surface

plasmon to increase the sensitivity to surface reactions. Because of the lower limits on SPR

detection, 0.1 to 0.2 nm effective thickness, imposed by the low signal-to-noise ratio the

increase in sensitivity that SPFS provides is a good alternative. SPFS is an ideal detection

technique for materials with a low packing density on the surface or for small molecules. The

intensity enhancement is due to the free electron gas in the metal that acts as a resonator that

is driven by the oscillation from the incoming laser beam. The intensity of the SP’s

evanescent field can be enhanced by a factor of 16 at the interface between the gold and the

aqueous medium.[9]

In order to use SPFS as a detection technique the material that is bound to the surface must

have a fluorescing molecule attached. The position of the chromophore with relation to the

gold surface can affect the fluorescence intensity, see figure 6. If the chromophore is close to

the metal surface the fluorescence is highly quenched by the dissipation of the energy into the

metal, figure 6 (a). Quenching refers to any process by which fluorescence intensity is lost

due to a transfer of energy. This phenomenon occurs at a distance of 5 to 10 nm from the

metal layer. From a distance of 10 to 20 nm, the excited surface plasmons transfer energy to

the chromophore, which upon de-excitation excite a red-shifted SP. At distances greater than

20 nm, the chromophore can freely emit photons. Up to a distance of a couple hundred

nanometers, the evanescent tail still enhances the intensity of the photon emission. By

placing the chromophore at a distance further than 20 nm from the gold surface, quenching

from the gold can be eliminated and the increase in intensity from the evanescent field can

still be utilized up to a distance of a couple hundred nm from the surface, see figure 7.[10]

9

Figure 6 shows a schematic of the fluorescence emitted by a dye molecule and the distance dependence of the fluorescence from the gold surface. (a) Fluorescence quenching due to the chromophore being within the Förster radius of 5-10 nm. (b) At a distance further from the gold surface the dye is excited by the surface plasmon and the fluorescence is coupled back into the metal exciting a lower-lying surface plasmon state. This is re-radiated at an angle corresponding to the dispersion curve. (c) At a distance still within the evanescent field, fluorescence emission is dominant.[10]

Figure 7: The fluorescence is highly quenched at distances smaller than 20 nm from the gold surface, dashed line. At distances greater than this the evanescent field can still be used to enhance the fluorescence signal, see solid line.[9]

The same experimental set-up that is used to perform SPR measurements, with a few

modifications, can be used for SPFS measurements. Therefore, it is possible to acquire

information on both the fluorescence and the surface plasmon resonance simultaneously.

10

3.2.1 Fluorescence The process of fluorescence occurs if an electron is excited to a higher energy state, usually

by a photon, but it can also be from heat, and then drops back down to the lower energy state.

The energy loss is in the form of a photon with energy EF, given in equation 15. The

wavelength of the emission depends on the magnitude of the energy of the transition.

FF hE ν= (15)

FFF E

chc==

νλ (16)

Fluorescence always starts from the lowest vibrational level of the excited energy state, but

can fall to any number of vibrational states in the ground state, figure 8. This means there can

be multiple wavelengths that are emitted as fluorescence, but usually the intensity of a

specific wavelength dominates.

Figure 8: A basic Jablonski diagram that shows the excitation and some emission paths between different energetic states. The energy being absorbed excites an electron to a higher energy level; energy is lost as heat during vibrational relaxation (VR). A photon can then be emitted as fluorescent light.

11

The lifetimes for the emission processes are significantly different. These can be seen in table

1. These differences determine the type of emission that will dominate under certain

circumstances. Vibrational relaxation and internal conversion occur at femto-second and

pico-second times. Whereas, fluorescence occurs at the nano-second time scale. In a

fluorescing system, because the vibrational relaxation is much faster, the fluorescence always

occurs from the lowest vibrational level in the excited state.

Type of Emission Rate Constant [s-1] Life Time [s]

Vibrational Relaxation (VR) 1011-1013 10-13-10-11

Internal Conversion (IC) 1011-1013 10-13-10-11

Intersystem Crossing 107-1010 10-10-10-8

Fluorescence 107-1010 10-10-10-8

Phosphorescence 10-1-104 10-4-10

Table 1: The processes of energy loss for a system excited by adsorption of a photon.

3.2.2 Experimental Set-Up The set-up used for these experiments included a He-Ne laser of wavelength λ=633 nm which

is coupled to the system via a high refractive index prism, LAFSN9 glass n=1.845 at a

wavelength λ=633 nm. A chopper intersected the beam; the chopper was connected to a lock-

in amplifier. Two polarizers were used to control the intensity of the incoming beam and to

insure that the incoming light was p-polarized. The flow cell was mounted on a goniometer

that would move at an angle θ. The reflected light was gathered by a lens and focused onto a

photodiode, which would move at an angle of 2θ. At the backside of the flow cell a photo-

multiplier tube (PMT) was used to collect the fluorescence from the sample. A lens was used

to focus the light onto the PMT and a filter was used to block the wavelengths of light that

were not of interest (central wavelength set at λ=670 nm, fwhh=10 nm). An attenuator (3 db)

was used in front of the PMT to decrease the signal to ensure that the intensity was not high

enough to overload the PMT. A shutter was used during the fluorescence experiments to

block the laser light at regular intervals during the experiment. The shutter allowed

experiments to be conducted for longer periods without the fluorophore bleaching. Bleaching

occurs if the high power from the laser light converts the chemical structure of the

12

fluorophore into another chemical that no longer fluoresces. All fluorescence experiments

were conducted in a black box to eliminate any outside source of photons.

Figure 9: (a) shows a schematic of the SPFS set-up. A normal SPR experiment could be conducted using the same set-up and not gathering data with the PMT. (b) The experiments were conducted using a cell containing an aqueous solution connected to the sample reservoir via Tygon tubing. The sample was continuously cycled through the cell using a peristaltic pump. [1]

All SPR experiments were done in-situ and in an aqueous environment. To accomplish this a

flow cell was used. A PDMS spacer with an ovular hole in the center was sandwiched

between the substrate and a quartz slide to create an empty chamber. Two tubes were

connected through holes to the chamber. The tubes were connected to a peristaltic pump by

Tygon tubing that continuously circulated the sample through the flow cell.

13

3.3 Neutron Reflectivity

Neutron reflectivity is a useful method for determining the surface structure of materials and

was used as a secondary experimental technique to compare with the results found by SPR

and SPFS. A neutron beam is used to irradiate a sample on a surface at an angle, θ, the

neutrons that interact with the nuclei of the material are scattered. The reflected neutrons are

collected by a detector and by analyzing the intensity of the collected neutrons as a function

of the wavevector ; structural information about the material can be determined. In neutron

reflectivity the one-dimensional scattering length density (SLD) is the important parameter

that is determined. The SLD gives a profile of the distribution of different materials on a

surface; thus, structural information can be obtained.

qr

The incident neutrons interact differently with each isotope or atom present in the material

being analyzed to give the SLD profile. Hydrogen and deuterium give very different SLD

profiles and this can be used to contrast different materials in a sample. For example, in the

experiment that was conducted for this thesis buffer containing deuterated water was used to

distinguish between the buffer and the deposited proteins containing hydrogen.

The information about the intensity of the reflected neutrons is obtained in relation to the

scattering vector, . qr qr has units of inverse length; the quantities that have units of inverse

length are often referred to as being in “q-space” as opposed to “real-space”. The scattering

vector can be determined by subtracting the incoming wave vector, 0kr

, from the outgoing

wave vector, k , see equation 17. The magnitude of the scattering vector is dependent on the

wavelength of the neutrons used, λ, and the scattering angle, θ. It can be calculated using

equation 18. A diagram of the relationship between scattering vector and wave vectors is

given in figure 10. [11]

r

0kkqrrr

−= (17)

=

2sin4 θ

λπq (18)

14

The magnitude of the scattering vector determines the scale of the features being probed. If

the features of interest are large, a small qr is needed. Vice versa, if the features of interest

are small, a large is needed. qr

Figure 10: The scattering vector is the result of subtracting the incoming wave vector from the outgoing wave vector.

In neutron reflectivity, the quantity that is measured in the experiments is the Fresnel

reflectivity, which is the square of the reflectance. The Fresnel reflectivity is scanned for a

region of -values and then plotted. For the most basic example where you have a thin film

with a uniform SLD on a substrate such as silicon, see figure 11, the curve that you obtain

will look like the example given in figure 12.

qr

Figure 11: A schematic of the incident and reflected neutron beams on the surface. The graph on the bottom shows the one-dimensional SLD profile, ρ(z), for the different materials.[12]

15

Figure 12: The simplest example of a scan that can be obtained for a thin film on a substrate by monitoring the Fresnel reflectivity for a range of qr -values.[12]

There are a number of film properties that will change the shape of the neutron reflectivity

curve. One of the most interesting for comparing the neutron reflectivity results to SPR is the

thickness of the thin film on the surface. The thickness can be approximated directly from the

reflectivity curve because it is related to the distance between the minimum values of the

oscillations seen in figure 12. The thickness is proportional to the inverse of the ∆k0, where

∆k0 is the distance between the troughs of the oscillations. The composition of the material

also has a direct effect on the SLD, which in turn affects the neutron reflectivity curve. The

value of the SLD affects the height of the oscillations. The height position of the reflectivity

curve in relation to the reflectivity curve of the substrate alone is determined by the value of

the SLD of the thin film in relation to the substrate.

The composition of the film and the thickness are important factors that influence the shape of

the neutron reflectivity curves, and they are the values that can be solved for by fitting the

obtained data. Nevertheless, they are not the only factors that influence the shape of the

reflectivity curve; the surface roughness and the absorption properties of the film also play a

16

large role in the reflectivity. For a more detailed look into these properties the reader is

referred to the review by Zhou et al. [12]

3.3.1 Data Analysis For the neutron scattering experiments conducted at a surface, the Surface Profile Analysis

Reflectometer (SPEAR) was used. The experiment was conducted at the Los Alamos

Neutron Science Center (LANSCE), in the United States. The neutron source at LANSCE is

a pulsed cold neutron source. The beam spot size was 8 mm by 30 mm.

Figure 13: A schematic of the neutron reflectivity experimental set-up. [13]

The SPEAR reflectometer is a time-of-flight (TOF) reflectometer. A basic sketch of the set-

up can be seen in figure 13. A neutron beam is incident to the sample at an angle of 0.9˚ to

the horizontal. The beam contains neutrons with a range of wavelengths from 1 to 32 Å. The

q-range that is created by these wavelengths is from 0.006 to 0.20 Å-1. The neutrons that are

scattered by the sample are collected by an array of detectors. [14]

The data was fit with a simple box model using the program Parratt32. [13] The program uses

the Parratt recurrence formula to calculate the wave functions for each of the layers

17

represented by the boxes. The boundary conditions for each layer are defined according to

the previous layer, the initial layer being the substrate. Because the substrate is usually silicon

and the properties are well known, the defining of the boundary conditions for the thin film

deposited can be defined and the thickness found. [12]

3.4 Materials and Methods

3.4.1 Streptavidin and Avidin Avidin and Streptavidin are very similar tetrameric glycoproteins with high binding affinities

for biotin, Ka ~ 1015 M-1. Each of the four monomers that compose avidin and streptavidin

can bind one molecule of biotin. Avidin is a common protein found in the whites of chicken

eggs; it has an isoelectric point of around 10.5 and its mass is 68,300 D, each subunit

weighing 18,000 D. [15] Avidin is positively charged in a neutral buffer and contains an

oligosaccharide component; because of this, it can nonspecifically bind with negatively

charged surfaces and with nucleic acids. Streptavidin, which is obtained from bacteria, does

not have this problem. SA has a mass of 52,800 D and an isoelectric point that is close to 7.

Streptavidin is commonly used to bind biotinylated molecules to a solid surface for biosensing

purposes. [16]

Figure 14: The structure of streptavidin. [17, 18]

The chromophore that was used to fluorescently label the streptavidin was Alexa Fluor 647.

The emission and absorption diagram for the dye can be see in figure 14. The emission peak

is at 650 nm, while the absorption peak occurs at 668 nm.

18

Figure 15: The emission and adsorption diagram for Alexa Fluor 647. [19]

3.4.2 Immunoglobulin G

Biotinylated goat anti-rabbit immunoglobulin G was also used in the experiments. IgG is a

large antibody, 156,000 D, which is commonly found in the human body, along with other

animals. [15] The B-IgG was purchased from Molecular Probes and was used as purchased;

the degree of biotin labeling was 5.0-5.2 biotin molecules per protein.

Figure 16: The structure of immunoglobulin G. [18]

Biotin is commonly known as vitamin H and is essential to metabolism and growth in

humans. It is a small molecule, 224.31 D, and can easily be attached to other molecules, e.g.

proteins and DNA oligomers. The biotinylated molecules can then be bound to a surface

using the specificity of the streptavidin-biotin complex. [20]

19

O

O

NHNH

S

OH

Figure 17: The structure of biotin.

3.4.3 Surface modification by NHS/EDC binding In these experiments, dithiodipropionic acid was used to prepare the gold surface for the

binding of the first protein layer. Because of the high binding affinity of the sulfur to gold

surfaces, thiols are an ideal molecule for further functionalization of surfaces. [21] The

chemical structure of this specific thiol is given in figure 18. The reaction of the dithiol is

given in equation 19; the sulfur-sulfur bond breaks to form two thiolates on the gold surface.

Figure 18: The structure of dithiodipropionic acid.

)(2)0(2 IAuSRAuRSSR −−→+−−− (19)

The thiolate on the gold surface consist of three carbons, with a carboxyl end group. Previous

studies have suggested that self-assembled monolayers of short-chained thiols do not form the

well-ordered layers that longer thiol molecules form. This is because the stability that the van

der Waals forces provide is not as strong in the shorter chains. [22]

The carboxyl end group on the thiol is ideal for using carbodiimide coupling with N-

Hydroxysuccinimide (NHS) and 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) to

bind an amino group on a protein to the gold surface. EDC forms an O-urea derivative which

by activating the carboxyl end groups on the thiolate. This reaction is shown in figure 19.

The derivative reacts with the NHS to form an intermediate; the amine group in the lysine

residues replaces the derivative. If the protein is added to the reaction vesicle, the amine

group on the protein replaces the NHS intermediate. Ethanolamine is added as the last step to

react with the sites that were not replaced with proteins. [23, 24]

20

Au

S

OH O

+EDC

H5C2

N

NH C

Au

S

O

CH3

CH3

N C3H6

+ O

O

OH

N

NHS

SA

+

C

Au

S

O

O ON

NH2

C

Au

S

O

HN

SA

Figure 19: A schematic of the covalent bonding of the first protein layer, in this case streptavidin. The dithiodipropionic acid is initially bound to the gold layer via sulfur bonding. This is followed by the activation of the carboxyl groups with N-Hydroxysuccinimide (NHS) and 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). The last two steps involve the binding of the streptavidin to the active sites, and finally the passivation of the unreacted site with ethanolamine (not shown.)

After the immobilization of the initial streptavidin, or avidin layer, biotinylated IgG molecules

can be bound to the surface via the high affinity for the streptavidin and biotin.

21

3.4.4 Surface modification by biotinylated thiol binding

A second deposition technique was used as a comparison for the NHS/EDC immobilization

technique. A thiol mixture of 11-mercapto-(8-biotinamido-4, 7, dioxaoctyl-)-

undecanoylamide and 1-mercapto-undecanole with a ration of 1:9 was self-assembled on the

gold surface. The SA was then directly bound to the biotin end groups. The structures of

these thiols are given in figure 20. This technique is well known and has been extensively

studied. [16, 25]

Figure 20: The chemical structure of 11-mercapto-(8-biotinamido-4, 7, dioxaoctyl-)-undecanoylamide, top, and 1-mercapto-undecanole, bottom.

22

3.5 Applications Many studies on biological multi-layer systems have been done with both the architecture and

the function in mind. There have been a couple of layer-by-layer systems that have been

developed for immunosensing purposes. [26] An example of two of these studies is an

alternating polyelectrolytes and charged colloids [27], and a system of alternating avidin and

biotinylated antibodies for the detection of glucose and lactate. [28]

There have also been many organic multi-layer systems that have been built using

electrostatic interactions, the most basic being alternating layers of polyelectrolytes with

opposite charges. [29] These polyelectrolyte multi-layer systems have been used to study the

energy transfer between chromophores. [30] Alternating polyelectrolytes and DNA layers

have also been used for studying dye interactions with DNA. [31] As the experiments

conducted for this thesis, biological affinity has also been used to build alternating layers of

biotinylated polymers and avidin. [32]

The multi-layer architecture used for these experiments is ideal for studying distance

dependence relationships at the nanometer scale. Because of the highly specific binding

between biotin and streptavidin, the multi-layer system is well defined. This could give it an

advantage over electrostatically bound systems. This system is well suited for studying some

fundamental aspects of surface plasmon enhanced fluorescence spectroscopy. This

architecture is currently being used for comparing aspects of fluorescence studied using the

Kretschmann prism coupled and the grating coupled surface plasmon resonance.

A second application of this multilayer architecture is to use it to boost the sensitivity of

biosensing applications that use chromophore labeled molecules as the species being detected.

Cui et al have shown in a similar system, that they can increase the binding ability for an

immunosensing detector with an alternating protein system. [33] The purely biological

composition of the multi-layer system is also an interesting property.

23

4 Procedure The substrates used in the experiments were LASFN9 glass slides. An Edwards evaporator

was used to apply a thin coating of gold, about 50 nm, onto one side of the glass slides.

Occasionally one nm of chromium was deposited before the gold to improve the adhesion of

the gold to the glass. Before deposition of the gold, the slides were thoroughly cleaned with a

2% solution of Helmanex and Milli-Q water. The gold-coated slides were then incubated

outside the flow cell over night in a solution of 1 mM 3,3’ dithiodipropionic acid in ethanol to

self-assemble the thiols on the gold surface. The slides were rinsed for half a minute with

ethanol and dried under a stream of nitrogen. The slide was then index matched, using oil

with the same refractive index as the glass slides and the prism, to the backside of the prism.

The slide and prism were then mounted to the flow cell.

The carboxyl groups on the thiols bound to the surface were then activated using NHS/EDC

chemistry for 15 minutes, see section 2.4.3 for details. A solution of 20 µg/ml streptavidin in

acetate buffer with a pH of 4.0 was then introduced to the flow cell and allowed to react for at

least 5 hours. In some experiments, the streptavidin (SA) was replaced by avidin or Alexa

Fluoro labeled streptavidin (AFSA). The surface was then rinsed with HEPES buffer. 1M

ethanolamine with HCl pH 8.5, pH adjusted with NaOH, was used to block the unreacted

carboxyl groups. The ethanolamine solution was circulated through the flow cell for 10

minutes and rinsed for 1 minute with HEPES buffer.

After deposition of the initial protein layer, a second protein solution of biotinylated

immunoglobulin G (B-IgG) in HEPES buffer was introduced to the flow cell and allowed to

react for half an hour. The surface was then rinsed with HEPES buffer for a full minute. The

following layer could be composed of one of the following: avidin, SA, or AFSA. Again, the

protein was introduced as a solution in HEPES buffer for thirty minutes. It was cycled

through the flow cell and then rinsed for a minute with HEPES buffer. These processes were

repeated until the desired thickness was achieved, the final product being alternating layers of

B-IgG and avidin, SA, or AFSA. A working concentration of 20 µg/ml was used for all

protein solutions unless otherwise noted. The protein solutions were prepared using a HEPES

buffer that contained 0.005% non-ionic surfactant to counter the loss of material due to the

proteins physisorption to the hydrophobic walls of the eppendorf tubes and the Tygon tubing.

24

The layer thickness and the fluorescence intensity before and after deposition steps were

investigated with SPR. The surface plasmon resonance was measured be collecting the

reflected laser beam with a photo diode. The fluorescence intensity was simultaneously

collected from the backside of the sample with a PMT. The maximum intensity of the

reflected laser beam was always set to 80% reflectivity.

The binding kinetics can be investigated by keeping the incident angle constant and observing

the change in the reflectance as a function of time, as the surface plasmon angle shifts. For all

experiments, the angle was set so that the initial reflectance was 25%. This was the chosen

value because it corresponds to the section of the scan where the curve is relatively linear, and

thus the change in reflectivity can be directly correlated to the change in thickness, assuming

that the refractive index does not change.

25

5 Results and Discussion 5.1 Thickness This experiment was conducted using the procedure described in section 4. The deposition

order was AFSA, B-IgG, SA, followed by B-IgG. This sequence was repeated four times,

table 2.

The thickness of each layer was calculated by fitting the surface plasmon scans using the

Winspall program. The fit parameters are given in table 2. The refractive index used to fit

the data for the protein layers was n=1.45. This refractive index was assumed to be similar

for all of the proteins and is commonly assumed for proteins studied by SPR. [16]

0 2 4 6 8 10 12 14 16 18

2468

101214161820222426283032343638

Layer

Thic

knes

s [n

m]

0

2

4 Change in S

PR

Angle [degree]

Figure 21: The thickness after each additional layer for and alternating SA and B-IgG architecture. The first layer deposited was avidin. The growth rate was found to be linear with a slope of 1.83 nm per layer.

The multi-layer system was deposited in a linear fashion; this can be seen in figures 21 and

22. The SPR minimum angle shift per layer was fit and the slope found to be 0.23 degrees per

layer. In thickness, this corresponds to 1.83 nm per layer if the SPR curves of both protein

layers were fit using a refractive index of 1.45.

26

0 50 100 150 200 250 300 350 400 450 500468

1012141618202224262830323436

Thic

knes

s [n

m]

Time [min]

Figure 22: The kinetics for the alternating SA and B-IgG multilayer system. The thickness obtained for the layers were linear in relation to the deposition step.

The SA layers had an average thickness of 1.8 nm. The dimensions of SA are known to be

4.5 x 4.5 x 5.3 nm from x-ray scattering. [34] The thickness of a SA monolayer bound to

biotinylated thiols has been extensively studied using SPR. This immobilization technique

gives a thickness of around 4 nm for the monolayer, which corresponds to a dense layer with

a packing density of 53%. [16] In contrast, the first monolayers bound with NHS and EDC

chemistry were found to be around 3 nm thick, which corresponds to a surface coverage of

about 40%.

27

Material

SPR Angle Shift (degree) ε-real ε-imm

Thickness(nm)

Total thickness (nm)

Prism 3.4036 0 0 Gold -12.751 2.1446 42.21 Thiol 2.25 0 1 1 1 AFSA 0.4 2.1025 0 2.9 3.9 2 IgG 0.5 2.1025 0 3.7 7.6 3 SA 0.3 2.1025 0 1.9 9.5 4 IgG 0.2 2.1025 0 1.9 11.4 5 AFSA 0.2 2.1025 0 1.6 13 6 IgG 0.3 2.1025 0 1.8 14.8 7 SA 0.2 2.1025 0 1.7 16.5 8 IgG 0.3 2.1025 0 2.1 18.6 9 AFSA 0.2 2.1025 0 1.6 20.2

10 IgG 0.2 2.1025 0 2.1 22.3 11 SA 0.3 2.1025 0 1.9 24.2 12 IgG 0.2 2.1025 0 2 26.2 13 AFSA 0.2 2.1025 0 1.3 27.5 14 IgG 0.2 2.1025 0 2.3 29.8 15 SA 0.3 2.1025 0 1.8 31.6 16 IgG 0.2 2.1025 0 1.8 33.4 17 AFSA 0.2 2.1025 0 1.7 35.1

Table 2: The thickness as calculated by fitting the SPR curves using the dielectric functions given in the table.

IgG with a molecular weight of 156,000 D is a protein that is three times larger than SA with

a molecular weight of 52,800 D. In theory, it should give a larger thickness than SA. In

general, this trend is seen. However, the difference is only slight; the average layer thickness

for SA was 1.8 nm and 2.2 nm for B-IgG. This can be accounted for by the fact that the SPR

curve fitting only gives an optical thickness. This means that information about the packing

density is also included in the thickness. The structure of the IgG is a large Y-shaped protein,

figure 14; whereas the SA molecule is quite compact. It would be expected that if the same

number of binding sites were available for each protein, different architectures would result.

The SA should give a compact layer, while the B-IgG layer would have a porous structure due

to the steric hindrances imposed by the arms of the antibody. A schematic of the proposed

binding structures is given in figure 23.

28

Figure 23: A schematic of the relative packing densities on a surface of streptavidin, top, and IgG, bottom. The molecule size ratios are not to scale. The IgG has relatively less dense packing due to the molecular structure.

Although in the previous example it is assumed that the proteins form a completely dense

monolayer on the surface, this is not the case. The first indication for this comes from the fact

that the proteins do not form layers with a thickness comparable to their dimensions. No

experiment was conducted to see how long the first SA deposition step would take to reach a

densely packed layer, but after four hours time a thickness of 2.9 nm was achieved. Due to

time constraints, it was decided that the density of the layers could be sacrificed for multi-

layer “height”. An experiment was conducted to determine the minimum time that could be

spent building each layer without sacrificing too much layer thickness. The first eight layers

of the experiment were built using an incubation time of one hour. The time for each layer for

the next eight layers was then halved. The results can be seen in figures 24 and 25 and table

3. The average thickness lost per bilayer was only 7.5 Å. Every experiment conducted after

this was conducted with each layer given half an hour to build.

29

0 2 4 6 8 10 12 140

1

2

3

4

5

Laye

r Thi

ckne

ss (n

m)

(2X-1) Layers of IgG-Biotin

60 min30 min

60 min30 min

Figure 24: The difference in thickness for streptavidin for a 60 minute and 30 minute immobilization period.

0 2 4 6 8 1 0 12 14 160

1

2

3

4

5

Laye

r Thi

ckne

ss (n

m)

2X Number of IgG-Biotin Layers

Figure 25: The difference in thickness for B-IgG for a 60 minute and 30 minute immobilization period. There was relatively little loss from switching to shorter deposition periods, so 30 minutes were used for the subsequent experiments.

30

Material

Angular shift

(degrees) ε-real ε-imm Thickness

(nm)

Distance from Au

(nm) Prism 3.4036 0 0 Chromium -1.2323 14.616 1.8 Gold -12.751 2.1446 39.2 Thiol 2.25 0 1 1 1 Av 0.3 2.25 0 2.7 3.7 2 IgG 0.6 2.1025 0 3.4 7.1 3 Av 0.3 2.1025 0 2.85 9.95 4 IgG 0.3 2.1025 0 2.46 12.41 5 Av 0.4 2.1025 0 2.74 15.15 6 IgG 0.3 2.1025 0 2.56 17.71 7 Av 0.4 2.1025 0 2.8 20.51 8 IgG 0.3 2.1025 0 2.62 23.13 9 Av 0.4 2.1025 0 2.63 25.76 10 IgG 0.3 2.1025 0 2.37 28.13 11 Av 0.3 2.1025 0 2.18 30.31 12 IgG 0.2 2.1025 0 2.25 32.56 13 Av 0.3 2.1025 0 2.15 34.71 14 IgG 0.3 2.1025 0 2.33 37.04 15 Av 0.3 2.1025 0 2.18 39.22 16 IgG 0.2 2.1025 0 2.37 41.59

Table 3: The thickness for and alternating avidin and B-IgG system found by fitting the SPR curves with the dielectric constants shown.

31

5.2 Structure model from Neutron reflectivity

A neutron reflectivity experiment was conducted at the LANSCE center in Los Alamos using

the equipment described in section 2.3.3. The reflectivity data was fit using the PARRAT32

program; the fitting parameters are given in table 4. The sample that was investigated with

neutron reflectivity was deposition of a SA layer immobilized on the 3,3’ dithiodipropionic

acid using NHS/EDC chemistry according to the procedure explained in section 4. The SA

layer was followed by a B-IgG layer. The reflectivity diagrams for all of the layers are shown

in figure 26.

0.0 0.1 0.2 0.3

1E-7

1E-6

1E-5

1E-4

1E-3

0.01

0.1

1

Ref

lect

ivity

Qz / Å-1

Au + Thiol Au + Thiol + SA Au + Thiol + SA + B-IgG

Figure 26: The neutron scattering results plotted as the Fresnel reflecivity versus the wavenumber in units of inverse angstroms. The experimental data is shown as the points and the lines are the fit curves.

Layer contents Thickness [Å] Scattering length

density [Å2] Roughness

[Å] Thiol 9 3.971E-6 14.4 Streptavidin 4 4.957E-6 27.8 B-IgG 4.6 4.519E-6 31.8

Table 4: The fitting parameters used to fit the data given in figure 25.

32

The thiol thickness was found to be 9 Å with a roughness of 14.4 Å. The roughness found for

the thiol layer could be compounded with the gold roughness. In thiol monolayers composed

of short alkane chains, it has been found that the layer thickness is smaller than expected due

to the high disordering. Larger alkane chains promote relatively ordered layers because of the

stabilizing effects of the van der Waals interactions between the carbon chains. For an alkane

thiol with three carbon groups the thickness was found to be about 5Å. [22] The carboxyl

groups will add another couple of angstroms to the thickness. [35] The value of 9Å with a

high degree of disorder is an acceptable value. From this experiment we were able to assume

a thickness of 1 nm for the fitting parameters in the SPR experiments.

The thickness found for the other two layers is suspected to be non-representative of the

layers built in the other experiment. Due to transportation problems, it is thought that the

protein solutions might have been damaged.

The thickness that was found for the first SA layer was 4 Å; this is a full magnitude of order

smaller than what is expected from a compact monolayer on a surface. The roughness of

almost 28 Å indicates that the layer is certainly not a compact monolayer. The high values for

the SLD indicate that there is a large amount of deuterated water present in the layer, which

also indicates a dilute protein coverage on the surface. The thickness and roughness of the

subsequent B-IgG layer, 4.6 Å and 31.8 Å respectively, also indicates that the system was

very dilute and formed very small layers. The large SLD for the B-IgG layer also indicates a

high amount of water molecules present in the layer, indicating that it was far from a packed

monolayer. These results are consistent with the results found in other experiments where a

failure due to errors in the procedure followed. An explanation of what might have happened

in this experiment is that very few molecules of SA were deposited in the first step and in the

following deposition of the B-IgG the proteins only bound to the few SA present on the

surface and a pillar structure was formed rather than the matrix that was expected. A

schematic of this is shown in figure 27.

33

Figure 27: A schematic of the proposed surface architecture for the neutron reflectivity experiment. The deposited layers were very dilute on the surface with the B-IgG probably only depositing on the dilute SA layers deposited in the first step forming a pillar structure.

34

4.3 Streptavidin vs. Avidin It has been shown that avidin and biotinylated IgG can form layer-by-layer like architectures.

[33] Streptavidin is a similar protein to avidin, but it has one large advantage over avidin, it is

less likely to non-specifically bind. Therefore, it is preferred in binding molecules to surfaces

as probes for biosensing applications.

In this experiment, two different multi-layer systems were built: the first with alternating

avidin and B-IgG, the second with SA and B-IgG. Both systems started with the same

thickness for the first layer, so both could be compared with the same starting architecture.

As can be seen in figure 28, both systems followed a linear thickness increase per layer

deposited.

-4 -2 0 2 4 6 8 10 12 14 16 18 20 22-5

0

5

10

15

20

25

30

35

40

45

50

55

Thic

knes

s [n

m]

Layer

Streptvidin and B-IgG Avidin and B-IgG

Figure 28: The thickness versus deposition step for two different architectures. The first layer for both systems was avidin. The thickness of the streptavidin archtecture, squares, was smaller than that of the avidin system, circles. The SA and B-IgG system had a growth rate of 1.8 nm per layer, as opposed to the avidin/B-IgG system with 2.5 nm per layer growth.

35

Alternating Streptavidin and

B-IgG layers Alternating Avidin and B-IgG

layers

Material Thickness

(nm)

Distance from Au

(nm) MaterialThickness

(nm)

Distance from Au

(nm) Prism 0 Prism 0 Gold 42.21 Gold 39.2 Thiol 1 1 Thiol 1 1

1 AFSA 2.9 3.9 Av 2.7 3.7 2 IgG 3.7 7.6 IgG 3.4 7.1 3 SA 1.9 9.5 Av 2.85 9.95 4 IgG 1.9 11.4 IgG 2.46 12.41 5 AFSA 1.6 13 Av 2.74 15.15 6 IgG 1.8 14.8 IgG 2.56 17.71 7 SA 1.7 16.5 SA 2.8 20.51 8 IgG 2.1 18.6 IgG 2.62 23.13 9 AFSA 1.6 20.2 Av 2.63 25.76 10 IgG 2.1 22.3 IgG 2.37 28.13 11 SA 1.9 24.2 Av 2.18 30.31 12 IgG 2 26.2 IgG 2.25 32.56 13 AFSA 1.3 27.5 Av 2.15 34.71 14 IgG 2.3 29.8 IgG 2.33 37.04 15 SA 1.8 31.6 Av 2.18 39.22 16 IgG 1.8 33.4 IgG 2.37 41.59 17 AFSA 1.7 35.1

Table 5: A comparison of the thickness for two systems: an alternating SA and B-IgG system and an alternating avidin and B-IgG system.

36

The slope of the line that was fit to the thickness graphs for the avidin with alternating B-IgG

layers was 2.5 nm per layer. The slope of the alternating SA and B-IgG system was 1.8 nm

per layer. This is a significant difference, which is not accounted for in the size difference in

the molecules. The mass of SA is 22% smaller than avidin, while the growth rate of the

avidin system was 25% larger than that of the SA system. The average thickness for the

avidin alone in the avidin system was 28% larger than the average SA thickness. This extra

growth in the avidin system could be attributed to some non-specific binding of the avidin

layers.

When the differences in the B-IgG growth for both systems were investigated, it was found

that the growth rate for the biotinylated IgG in the avidin system had an average thickness that

was 13% larger than the B-IgG for the streptavidin system. This most likely has to do with

the mechanics of growth, if there are less binding sites available in the SA system due to a

lower mass of SA depositing in the previous layer it is only logical that there will be less

binding from the B-IgG layer.

37

5.4 Experimental reproducibility Two experiments were conducted in order to compare the reproducibility of the multi-layer

system over a large number of layers. The first SA layers of the two experiments were both

deposited with the NHS/EDC chemistry using the procedure described in section 4.1.

However, one change in the procedure was made. In experiment I, acetate buffer pH 5.0 was

used as opposed to pH 4.0 which was used in experiment II.

Because the isolelectric point of SA is around seven, it has no charge in HEPES buffer pH

7.4. Acetate buffer with a lower pH was used to introduce charges to the protein. If pH 4.0

was used instead of pH 5.0, more charges were introduced to the protein. This increases the

binding efficiency because the positive charges on the protein will be attracted to the

negatively charged gold surface. The results of this effect can be seen in table 5. The

thickness of the SA layer in experiment I was 1.8 nm compared to 2.9 for experiment II; this

is a difference of 40%.

-6 -4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24

-5

0

5

10

15

20

25

30

35

40

45

50

I: SA and B-IgG, Acetate buffer ph 5.0 II: SA and B-IgG, Acetate buffer pH 4.0

Thic

knes

s [n

m]

Layer

Figure 29: The thickness for two similar systems. Both were alternating SA and B-IgG, with the exception of the first layer. One experiment was conducted with the first layer being deposited in acetate buffer pH 5.0, circles, and the second in acetate buffer pH 4.0, squares.

38

Although the first layers had a different thickness, figure 29 shows that the growth rates were

the same for both experiments. The growth rate was found to be 1.8 nm per layer for both

experiments. The average SA thickness was comparable for both systems; 1.6 nm for

experiment I and 1.7 for experiment II. The B-IgG average thickness was also comparable at

2.1 nm for I and 2 nm for II.

I:Alternating Streptavidin and B-IgG layers

II: Alternating Streptavidin and B-IgG layers

MaterialThickness

(nm)

Distance from Au

(nm) MaterialThickness

(nm)

Total thickness

(nm) Prism 0 Prism 0 Gold 47 Gold 42.21 Thiol 1.0 1.0 Thiol 1 1

1 AFSA 1.8 2.8 AFSA 2.9 3.9 2 IgG 2.7 5.5 IgG 3.7 7.6 3 SA 1.9 7.4 SA 1.9 9.5 4 IgG 2.2 9.6 IgG 1.9 11.4 5 AFSA 1.6 11.2 AFSA 1.6 13 6 IgG 1.7 12.9 IgG 1.8 14.8 7 SA 2.0 14.9 SA 1.7 16.5 8 IgG 2.1 17.0 IgG 2.1 18.6 9 SA 1.8 18.8 AFSA 1.6 20.2 10 IgG 2.1 20.9 IgG 2.1 22.3 11 SA 1.8 22.7 SA 1.9 24.2 12 IgG 2.1 24.8 IgG 2 26.2 13 SA 1.9 26.7 AFSA 1.3 27.5 14 IgG 1.8 28.5 IgG 2.3 29.8 15 SA 1.5 30.0 SA 1.8 31.6 16 IgG 2.4 32.4 IgG 1.8 33.4 17 SA 1.1 33.5 AFSA 1.7 35.1 18 IgG 2.1 35.6 19 SA 1.0 36.6

Table 6: A comparison of the layer thickness for two experiments with the same procedure used, with the exception that the pH of the buffer for the first SA layer was different.

39

5.5 Multi-layer architecture’s stability against free biotin An experiment was conducted to test the stability of the protein multi-layer system against

free biotin. The theory being that the biotin might be able to replace the B-IgG if the protein

layers were not tightly bound to each other. In theory, the biotin could kick out the B-IgG and

the high binding affinity of the biotin molecules would block the available sights on the SA

and render the layer unable to build subsequent layers. If the layers were unable to withstand

the attack from the free biotin then a loss of material in the form of a shift in the surface

plasmon minimum to a smaller angle would be observed.

Four bilayers of alternating SA and B-IgG were deposited and then pure biotin was added to

the flow cell. As figure 30 shows, there was no gain or loss in material. The surface plasmon

resonance angle before, squares, and after, circles, stays constant. The kinetic curve that is

shown in figure 31 also shows this. Although there is a small decrease in percent reflectivity

at the beginning of the experiment, it is most likely due to changes in refractive index between

the pure buffer and the biotin solution. This is also true for the jump in the kinetic curve at

shortly after the rinsing with buffer, the arrow indicates when the buffer was used to rinse.

45 50 55 60 65

0

20

40

60

80 Before addition of Biotin After addition of Biotin

Ref

lect

ance

[%]

Angle [degree]

Figure 30: The surface plasmons scans conducted before and after addition of pure biotin confirm that there was no gain or loss of material.

40

0 5 10 15 20 2524,00

24,25

24,50

24,75

25,00

25,25

25,50

25,75

26,00

Ref

lect

ance

[%]

Time [min]

Figure 31: The kinetics of the addition of free biotin to the multilayer system.

41

5.6 Streptavidin immobilization techniques An experiment was conducted to compare the binding method for the first streptavidin layer

used for the other experiments in this thesis and a second commonly used immobilization

technique. A mixture of 11-mercapto-(8-biotinamido-4, 7, dioxaoctyl-)-undecanoylamide,

which will be called “B-thiol,” a biotinylkated thiol and 1-mercapto-undecanole. 1-mercapto-

undecanole dilutes the B-thiol on the surface to maximize the binding efficiency of the SA to

the surface; it will be called the “spacer” thiol.

Both experiments built a multi-layer architecture, with only slight differences. The angular

shifts for the SPR curves can be seen in figures 32 and 33.

54 55 56 57 58 59 60

0

5

10

15

20

25

30

35

45 50 55 60 65

0

20

40

60

80

Ref

lect

ance

[%]

Angle [degree]

Ref

lect

ance

[%]

Angle [degree]

3,3' Dithiodipropionic acid SA B-IgG

Figure 32: SPR curves for alternating SA and B-IgG layers. The first layer was immobilized using NHS/EDC chemistry. The first curve is of dithiodipropionic acid on gold.

42

54 55 56 57 58 59 60

0

5

10

15

20

25

30

35

45 50 55 60 65

0

10

20

30

40

50

60

70

80

90

Ref

lect

ance

[%]

Angle [degree]

B-Thiol and spacer SA B-IgG

Ref

lect

ance

[%]

Angle [degree]

Figure 33: The SPR curves for alternating Sa and B-IgG. The first SA layer was immobilized using biotinylated thiols self assembled on a gold surface. The first curve is for the thiol on gold.

The first very noticeable difference is in the thickness of the first SA layer. For the SA bound

using NHS/EDC chemistry the thickness was 1.8 nm, for the biotinylated thiol system the

thickness was 2.9 nm. This is about one nm smaller than the normal thickness achieved using

this method, but it is still a difference of 40%.

The second difference is the thickness of the last four layers. In the NHS/EDC

immobilization the growth rate is 0.3 nm per layer larger than the growth rate for the B-thiol

immobilization technique. Both the SA and the B-IgG layers were smaller in the B-thiol

system. For the B-IgG, the average thickness was 5% smaller for the B-thiol system and the

SA average thickness was 27% smaller.

The overall thickness for the B-thiol system, 15.4 nm, was 16% larger than the overall

thickness for the NHS/EDC system after six layers. One reason for this is that the thickness

of the B-thiol and spacer is larger than the 3,3’ dithiodipropionic acid, 12.9 nm. The other

contribution comes from the thickness of the first SA and the first B-IgG layers. The B-thiol

immobilization gives a very large thickness for the first SA, as was discussed earlier. In

addition, the following biotinylated IgG layer is also much larger, 42%.

43

NHS/EDC chemistry for binding of

first SA layer Biotinylated thiol for binding of first

SA layer

Material Thickness

(nm) Distance from

Au (nm) Material Thickness

(nm) Distance from

Au (nm) Prism 0 Prism 0 Gold 47 Gold 47 Thiol 1.0 1.0 B-Thiol 1.5 1.5 1 AFSA 1.8 2.8 SA 2.9 4.4 2 IgG 2.7 5.5 IgG 4.7 9.1 3 SA 1.9 7.4 SA 1.4 10.5 4 IgG 2.2 9.6 IgG 2.0 12.5 5 AFSA 1.6 11.2 SA 1.2 13.7 6 IgG 1.7 12.9 IgG 1.7 15.4

Table 7: A comparison of the layer thickness obtained from two different immobilization schemes for the first SA layer.

1 2 3 4 5 62

4

6

8

10

12

14

16

NHS/EDC binding B-thiol binding

Dis

tanc

e fro

m A

u [n

m]

Layer

Figure 34: The thickness difference for systems for which the first SA layer was bound using the NHS/EDC chemistry, squares, and B-thiol self-assembled on gold, circles.

44

5.7 Thickness effects of Alexa Fluoro The average thickness of the SA and the Alexa Fluoro labeled SA was 1.8 nm for both

systems. The Alexa Fluoro was assumed to have no effect on the binding of the proteins. In

all experiments in which AFSA was used instead of SA they were treated the same. The

thickness of the B-IgG that was deposited after the AFSA and SA were also compared and no

discernable difference was found. The thickness data that was used for these comparisons can

be found in table 2.

45

5.8 Fluorescence Effects of Alexa Fluoro Streptavidin labeled with a chromophore was used in some of the experiments in place of

normal SA. A system of AFSA, B-IgG, SA, and B-IgG protein layer deposition steps were

repeated four times. The kinetics and SP scans were monitored using both the photodiode to

detect reflected light from the prism and a PMT was used to gather fluorescence light from

the backside of the flow cell. A 3-db attenuator was used in this experiment to protect the

PMT from the high intensity light from the multiple chromophores deposited. The thickness

results for this experiment were discussed previously in section 4.1. The thickness

information can be found in table 2.

As can be seen in figure 35, the fluorescence intensity increases with every additional AFSA

layer. For the experiments the layers were built sequentially on top of each other, so the

bottom layers containing chromophores also contribute to the fluorescence signal detected.

Nevertheless, if the fluorescence contribution for the layer deposited directly before the AFSA

is subtracted out, figure 34, it can be seen that the fluorescence intensity still increases for

each additional chromophore labeled SA deposited. The chromophores could be used to

probe the evanescent field, as was done previously. [36] As can be seen in the graph of the

normalized fluorescence intensity versus distance from the gold surface, figure 36, the results

are not similar to what has been previously obtained. Vasilev et al. have shown that the

fluorescence signal versus the distance from the gold surface follows a S-shaped curve. The

fluorescence signal is significantly quenched at distances close to the surface and at larger

distances the signal levels out due to the decrease in excitation due to a decrease in the

evanescent field intensity. The results from this experiment show that there is almost a linear

relationship between the distance from the metal surface and the intensity of the fluorescence

signal. This deviation from the previous results is probably due to the convolution of the

fluorescence intensity with the size of the proteins. The proteins are labeled with 4 to 5

chromophore molecules per protein and with the protein diameters in the range of 4 nm, the

chromophores are not placed at a specific point, but rather at a range of distances.

Figure 35 also shows the angular shift of the maximum fluorescence intensity with the shift of

the SPR. This is to be expected and the can be explained by the fact that the SP used to

excited the chromophore and the fluorescence intensity peak shifts accordingly.

46

44 46 48 50 52 54 56 58 60 62 64 66 68

0

20

40

60

80

0

5000

10000

15000

20000

25000

30000

35000

Ref

lect

ance

[%]

Fluo

resc

ence

[cps

]

Angle [degree]

Figure 35: The surface plasmon scans and the corresponding fluorescence intensities for a multilayer system with alternating AFSA, B-IgG, SA, and B-IgG layers. Each of the five scans was taken after deposition of an AFSA layer.

5 10 15 20 25 30 35 40

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

1,1

Fluo

resc

ence

, nor

mal

ized

Thickness [nm]

Figure 36: The fluorescence versus the distance from the gold surface for 18 depositions steps.

47

An interesting effect was observed if additional layers of non-labeled dielectric material were

deposited on top of the dye labeled SA. If three additional non-labeled protein layers were

deposited on top of the AFSA layer closest to the gold surface, the fluorescence increased for

each additional layer, figure 37 (a) and (b) show this increase in fluorescence intensity.

Some theories of why the fluorescence intensity increases with additional dielectric material

have been proposed. [37] The first is that there is a change in the conformation of the first

AFSA layer due to the deposition of additional layers. The second reason could be due to a

change in the SP electro-magnetic field distribution. Simulations were done using the

Wingitter program to calculate the change in the magnetic field distribution upon addition of a

dielectric medium. The intensity of the magnetic field at a specific distance, in this case the

position of the chromophore layer calculated using the Winspall program, was simulated for

each of the additional protein layers, figure 38. The magnetic field intensities that were

calculated were modified using the Snell equation to account for the prism because the

Wingitter program was written for a different experimental configuration that did not include

a prism. In figure 38 the units of the magnetic field are given as A.U., which stand for

“arbitrary units”, it is still not clear what the intensity units are and what the true magnitude is.

These simulations were only used to give an idea of what the trend is for the intensity and to

compare this trend with the fluorescence intensity obtained.

As can be seen in figure 38, there is no increase in the maximum intensity of the H-field with

additional material added, but only an angular shift of the max intensity that corresponds to

the angular shift of the SP. This indicates that a change in the magnetic field intensity is not

the factor that leads to the increase in the fluorescence intensity with the addition of non-

absorbing material.

48

44 46 48 50 52 54 56 58 60 62 64

0

20

40

60

80

Angle [degree]

Ref

lect

ance

[%]

0

100

200

300

400

500

600

700

800

900

Fluorescence [cps]

1234

(a)

1,0 1,5 2,0 2,5 3,0 3,5 4,0600

650

700

750

800

850

Fluo

resc

ence

[cou

nts]

Layer

(b)

Figure 37: (a) The fluorescence intensity after additional layers of dielectric material were deposited onto an initial layer containing dye molecules, layer 1. (b) The fluorescence intensity increases with the addition of material.

49

1 2 3 4

44 46 48 50 52 54 56 58 60 62 64

0

20

40

60

80

Angle [degree]

Ref

lect

ance

[%]

0

1

2

3

4

5

6

7

Hy -Field Intensity [A

.U.]

Figure 38: Simulations for the magnetic field intensity at the position of the chromophore layer as calculated with Winspall. There is no increase in the magnetic field intensity with the addition of material that is more non-absorbing, layers 2, 3, and 4.

If a second AFSA layer was deposited about 15 nm from the metal surface, layer 5 in figure

39, the fluorescence intensity per additional protein layer decreased. This is what is to be

expected and the loss in intensity is most likely due to photo-bleaching effects associated with

exposure of the dye molecules to the laser light. Even with use of a shutter to shorten the

length of exposure time, the molecules were exposed to the laser light for around 3 to 5

minutes.

50

44 46 48 50 52 54 56 58 60 62 64 66

0

20

40

60

80

Angle [degree]

Ref

lect

ance

[%]

0

1000

2000

3000

4000

5000

6000

Fluorescence [cps]5678

(a)

Figure 39: The fluorescence intensity after additional layers of dielectric material, layers 6,7, and 8, were deposited onto an second layer containing dye molecules, layer 5.

4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0

4600

4800

5000

5200

5400

Fluo

resc

ence

[cou

nts]

layer

(b)

Figure 40: The fluorescence intensity decreases with the addition of material when additional non-absorbing material was placed on top of a second AFSA layer.

51

Vaselev et al. have also shown that when a dye molecule is placed closed to the metal surface

the photo-bleaching effects are lower than if the dye molecule is further from the surface. [38]

This could explain why the fluorescence intensity associated with layer 5 decreases with the

addition of more protein layers. However, this does not explain the increase in intensity for

the first AFSA layer with additional material added on top. As previously explained, this

could be due to additional effects from conformational changes of the protein layers that

contain the chromophores.

In general, many factors influence the fluorescence intensity. These include: the Foerster

quenching, the evanescent decay of the electro-magnetic field, and bleaching effects

associated with chemical changes in the dye from the energy absorbed by the chromophore

molecules. Further experiments would need to be done to separate these effects and to

explain the results of the experiment fully. Specifically, a second experiment would need to

be done without the second AFSA layer to separate the effects of the dye placement on the

gold surface from the effects of depositing additional dielectric material.

5 6 7 8

44 46 48 50 52 54 56 58 60 62 64 66

0

20

40

60

80

Angle [degree]

Ref

lect

ance

[%]

0

1

2

3

4

5

6

Hy -Field Intensity [A

.U.]

Figure 41: Simulations for the magnetic field intensity at the position of the second chromophore layer 5 as calculated using Winspall. There is no change in the magnetic field intensity with the addition of material that is more non-absorbing, layers 6,7, and 8.

52

5.9 Polymer and Protein Multi-layers A second multi-layer system was developed to see if thicker layers could be deposited with an

alternating polymer and SA system. A commercially available kit was used to biotinylate a

Polyethylenimine with an average molecular weight of 750,000. The biotinylation was

preformed according to a method previously described by Anzai et al.[32] These biotinylated

polymers (B-PEI) were then attached to the surface using the method described in section

3.4.4. A biotinylated thiol in a solution containing thiols with a carboxy end group were used

to attach a monolayer of SA to the surface. A solution of 1 µg/ml B-PEI in HEPES buffer

was then circulated through the flow cell for 30 minutes, and a second layer of SA was then

deposited. This process was repeated until the desired number of layers had been achieved.

A preliminary experiment was conducted to see if the polymers would build a multi-layer

system. As can be seen in figure 42, the shifts in the resonance curves show that there is a

significant amount of SA deposited after each B-PEI layer. Table 8 contains the thickness

information found by fitting the SPR curves with the Winspall program. The average

thickness for the SA layers was found to be 5.8 nm per layer. This is more than 3 times what

the average thickness for the Sa layers in the alternating Sa and B-IgG system was found to

be. The B-PEI was found to have an average thickness of only 1.1 nm per layer. This is

significantly less than was expected for a polymer with such a larger molecular weight. This

deviation from the expected results can be attributed to the fact that the polymer most likely

spreads across the SA monolayer rather than forming a thick layer. However, the polymer

does form a 3-dimensional matrix, which is what allows for such a large binding of the SA

molecules. A schematic for the proposed layer morphology is given in figure 43.

The thickness for the entire 17 layers that were deposited was found to be 62.3 nm thick

including the thiol layer; this corresponds to an angular shift of 7.7 degrees. When compared

with the alternating Sa and B-IgG system this is almost double the thickness achieved for the

same number of layers, 35.1 nm or 4.4-degree angular shift.

53

50 52 54 56 58 60 62 64 660

1

2

3

4

5

Thiol SA B-PEI

44 46 48 50 52 54 56 58 60 62 64 66 68 70-5

0

5

10

15

20

25

30

35

40

45

Ref

lect

ance

[%]

Angle [degree]

Ref

lect

ance

[%]

Angle [degree]

Figure 42: The surface plasmon resonance curves for 17 deposition steps of alternating B-PEI and SA. There was a total angular shift of 7.7 degrees from the first thiol layer to the last SA layer.

54

Material

SPR Angle Shift (degree) ε-real ε-imm

Thickness(nm)

Total thickness (nm)

Prism 3.4036 0 0 Gold -12.751 2.1446 42.21 Thiol 2.25 0 1.5 1.5 1 SA 0.35 2.1025 0 2.8 4.3 2 B-PEI 0.25 2.25 0 1.3 5.6 3 SA 0.85 2.1025 0 7 12.6 4 B-PEI 0.3 2.25 0 1.2 13.8 5 SA 0.9 2.1025 0 6.2 20 6 B-PEI 0.2 2.25 0 1.1 21.1 7 SA 0.85 2.1025 0 6.6 27.7 8 B-PEI 0.3 2.25 0 1.1 28.8 9 SA 0.65 2.1025 0 7 35.8

10 B-PEI 0.3 2.25 0 1 36.8 11 SA 0.55 2.1025 0 5 41.8 12 B-PEI 0.2 2.25 0 1.1 42.9 13 SA 0.65 2.1025 0 5.4 48.3 14 B-PEI 0.1 2.25 0 1 49.3 15 SA 0.6 2.1025 0 6 55.3 16 B-PEI 0.2 2.25 0 1 56.3 17 SA 0.45 2.1025 0 6 62.3

Table 8: The thickness values found by fitting the angular shift of the SPR using the Winspall program. The total thickness for 8.5 bilayers was found to be 62.3 nm.

Figure 43: A schematic of an alternating biotinylated PEI system. This system depicted with the polymer layers could be used to deposit a labeled streptavidin layer that would be better defined than in the system currently in use it could also push the sensitivity limits by providing a high density of binding sites.

55

6 Future Work

The neutron reflectometry experiments should be repeated in order to obtain a more accurate

picture of the structure of the multi-layer systems. The experiment should be repeated using a

procedure that is as close as possible to the procedure used in the SPR experiments, including

a flow system to reproduce the conditions used for these experiments.

The biotinylated PEI system should be characterized in more detail. Experiments should be

conducted using fluorescence molecules to compare the results to those found for the pure

protein system. This will not produce a system where the dye molecules are located at a

distinct distance from the gold layer. However, it will be an interesting system for comparing

the other extreme where the dye molecules are spread over a range of distances. This should

give some insight into the results obtained in section 5.8.

A second interesting experiment would be to change the ionic concentration in the buffer

solution. Because the PEI is a polyelectrolyte it should change its conformation under

different ionic conditions. When the ionic strength is low, the charges on the PEI molecules

will be unshielded and the polyelectrolyte will have a stretched conformation. There should

be more biotin available for binding of SA molecules, which would also increase the total

thickness. This experiment could be conducted using fluorescently labeled proteins to

investigate the changes in morphology in the films.

It would also be of interest to study the increase in sensitivity associated with the increase in

the probe binding sites that are available when using the matrix like system created by the B-

PEI multi-layer systems. The signal intensity of a target with a low concentration in solution

of this system could be compared with a conventional biosensor with a comparable thickness

it could also be compared with the protein multi-layer system used in these experiments.

A final experiment of interest would be to investigate the B-PEI films with neutron

reflectivity to compare the morphology of the pure protein multi-layers with that of the

polymer and protein multi-layer system.

56

7 Conclusion The multi-layer architectures composed of alternating SA and B-IgG used in this thesis were

found to build with a linear growth rate. The thickness of the layers was investigated using

neutron scattering and SPR. Experiments were also conducted to investigate the

reproducibility and the stability of the multi-layers; both experiments were found to be

successful. Two consecutive experiments conducted using nearly the same procedure were

compared to test the reproducibility and the growth rates for both experiments were found to

be 1.8 nm per layer. The stability test conducted using the addition of pure biotin to the

multi-layer system was conducted by comparing the SPR scans prior and after addition of free

biotin; there was no loss or gain of material, indicating that the multi-layer architecture is

stable.

The effects of using SA versus avidin to build the multi-layer system were studied using SPR.

The average thickness of the avidin layers was found to be 28% larger than the average

thickness of the SA. This extra growth in the avidin system could be attributed to some non-

specific binding of the avidin layers.

Two immobilization techniques for the attachment of the first SA layer to the metal surface

were compared with SPR; the first using covalent bonding between the SA and a thiol

derivative self-assembled to the surface, the second technique used the binding affinity of SA

and biotin to bind the SA directly to a biotinylated thiol on the gold surface. Both techniques

were found to be successful. For the SA bound using NHS/EDC chemistry the thickness was

1.8 nm, for the biotinylated thiol system the thickness was 2.9 nm, a difference of 40%.

Using SPR the thickness of Alexa Fluoro labeled SA as compared with unlabeled SA was

investigated; no difference in thickness was found. The fluorescence intensity of AFSA was

investigated using SPFS and found to increase the farther from the gold surface the AFSA

was placed; these results do not reproduce the results found using a different detection

technique, this is probably due to the convolution of the fluorescence intensity with the size of

the proteins. The angular shift of the maximum fluorescence intensity with the shift of the

SPR; this was expected.

57

The effect of adding additional non-labeled protein layers to a layer of AFSA on the

fluorescence intensity was also investigated using SPFS. If the AFSA layer was placed close

to the gold layer, the addition of dielectric material on top resulted in an increase in the

fluorescence intensity with each additional layer. This effect could be attributed to a change

in the conformation of the first AFSA layer due to the deposition of additional layers. If a

second AFSA layer was placed at around 15 nm from the metal surface the fluorescence

intensity was found to decrease due to bleaching effects.

Preliminary experiments were conducted to investigate the thickness of an alternating

biotinylated polyelectrolyte and SA system. The system was found to build bilayers that were

almost twice as thick as the protein multi-layer system used in these experiments. The B-PEI

and SA system looks like a promising architecture for further studies with SPR, SPFS and

neutron reflectivity.

58

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60

Acknowledgments

I would like to offer my thanks to Professor Wolfgang Knoll for allowing me to do my

masters work in his group. I would also like to thank the Physical Chemistry department at

the University of Mainz for allowing me to attend the Chemistry of Materials program.

Thank you to Dr. Fang Yu for all of the help getting started on this project and for all the help

clarifying all my questions.

I would also like to extend a big thank you to Dr. Ingo Köper for performing the neutron

reflectivity experiments and for fitting the curves.

Thank you to all of the Knollies for making being such a great group with wonderful

dynamics that made my work so enjoyable. I would also like to offer a special thanks to the

“office that never sleeps” for making the atmosphere in the office so conducive to working.

Last of all, I would like to thank my family and friends for the emotional support that I have

received throughout my studies.

61