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2017-01-25
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1Biotechnical Physics
Plasmonic Biosensors
2017-01-25
Lecture 2/2Andreas B. Dahlin
http://www.adahlin.com/
2017-01-25 Biotechnical Physics 2
Outline
About plasmonic biosensors:
• What is a surface-based biosensor?
• Plasmons in nanoparticles and on surfaces.
• Examples of plasmonics in biotechnology.
The connection to binding kinetics to surfaces: We can now measure Γ!
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One definition can be found in the handbook from the International Union for Pure and
Applied Chemistry (IUPAC):
A device that uses specific biochemical reactions mediated by isolated enzymes,
immunosystems, tissues, organelles or whole cells to detect chemical compounds usually
by electrical, thermal or optical signals.
2017-01-25 Biotechnical Physics 3
What is a Biosensor?
Ysignal
recognition element
(biological)
analyte
(biological or not)
Y”transducer”
The use of recognition elements
(or receptors) is sometimes
referred to as ”affinity based”
biosensing.
Not many! New biosensor technologies are more common in research environments.
Glucose sensor changed the life of diabetes patients. By some considered to be the only
truly successful biosensor and still developing. Quantitative!
Pregnancy tests (a lateral flow assay) detect human chorionic gonadotropin from urine.
(Also for ovulation.) Qualitative!
2017-01-25 Biotechnical Physics 4
Biosensors in Everyday Life
Clearblue
http://www.clearblue.com/
~10 nm Au
Yao et al.
Biosensors and Bioelectronics 2011
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Most sensors, not the least biosensors, need to calibrated. In a calibration experiment the
response to known doses of the variable of interest is measured.
The sensitivity, dynamic range and detection limit are defined from the calibration curve.
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Sensor Terminology
sen
sor
resp
on
se
(measu
red
)
environmental variable
(controlled)
slope = sensitivity
detection limit
dynamic range
zero
response
Most biosensors need to operate label-free to
be useful. This means that they work even if
the analyte does not carry any artificial label.
When operating label-free, the biggest
problems in biosensor technology is arguably
false positive results.
When we search for analytes (fish) in
biological samples we will always have a lot
of other molecules (fishes) present that can
interfere with the detection.
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The Challenge of Specificity
signal
transduction
target (analyte)
sample solution
bait (probe, receptor,
recognition element)
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Detect anything that binds to a surface! Several instruments exist, most are based on
optical measurements, some mechanical techniques and also a few other.
In this course you will learn about two techniques: Surface Plasmon Resonance (SPR)
and Quartz Crystal Microbalance (QCM).
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Surface Sensitive Techniques
Qsense (Biolin Scientific)
http://www.qsense.com/Biacore (GE Healthcare)
http://www.gelifesciences.com/
2017-01-25 Biotechnical Physics 8
Know Your Techniques!
You will learn the physics behind the techniques. The instruments will not be “black
boxes” to you when you encounter them in the future!
Sometimes things go wrong when scientists use machines they know nothing about…
Rich & Myszka
Journal of Molecular Recognition 2011
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The surface must be chemically
functionalized such that only the
analyte binds.
The techniques work in real-time,
which gives information about
binding kinetics.
Optical techniques like SPR often
enable multiplexing , detection of
multiple targets, by imaging mode.
2017-01-25 Biotechnical Physics 9
Surface-Based Detection
Y Y Y Y
other molecules binding
to recognition element
Y Y Y Y
Y Y Y Yanalyte
recognition element
functionalization
specific interaction
sample solution
sensor surface
inert background
Y
Y Y Y Y
Y Y Y Y
analyte binding to surface
other molecules binding
to surface
2017-01-25 Biotechnical Physics 10
Biosensor Scenarios
In vivo: Inside the living organism. Not for plasmonic biosensors!
In situ: Biological sample analyzed in artificial setting. Possible but difficult!
In vitro: Artificial setting, bottom-up synthetic biology. Perfect!
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Implications from Binding Kinetics
Remember the models for binding kinetics to surfaces! Let the A molecules represent
receptors and the B molecules targets (analytes). A + B → AB
If the surface is a sensor that gives a measurable response proportional to Γ:
• Real-time operation needed to determine kon and koff and to confirm equilibrium.
Measuring the equilibrium coverage gives only KD from C0 or vice versa.
• Diffusion models can estimate performance limits: A molecule can diffuse to the sensor
surface without binding to it, but not bind to the surface without first diffusing to it.
• Flow is often needed to prevent diffusion effects and reach Langmuir behavior.
• Small sensors give a higher diffusive flux due to edge effects. But the capture efficiency
is poor under flow (many molecules will be lost).
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What is a Plasmon?
Physical understanding: Plasmons are
collective oscillations in the free electrons
of metals.
Mathematical understanding: Plasmons
are solutions to Maxwell’s equations for
certain metal-dielectric geometries.
For a metal nanoparticle, the polarization
enhanced at certain frequencies of light.
For noble metals, this occurs for visible
light, which gives strong colors.
The Lycurgus Cup is the oldest example
(year ~400) of this kind of staining.
Ag, Au, Cu (60%, 30%, 10%) in glass
Freestone et al.
Gold Bulletin 2007
ambient light light inside cup
British Museum
http://www.britishmuseum.org/
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Absorption and Scattering
The polarizability (α) of the particle determines the absorption and scattering cross
sections (σ). The extinction is the sum of absorption and scattering.
For gold nanoparticles (small and spherical):
Blue light is absorbed (true for all gold).
Green light is absorbed and scattered (by the plasmon).
Red light is transmitted (low extinction).
αkσ Imext
24
scaπ6α
kσ
scaextabs σσσ
British Biocell International
http://www.bbisolutions.com/
Here k is the incident wavevector (k = 2π/λ) and α is
defined as a volume. The cross sections are areas
(shadows)!
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Electrostatic Approximation
By considering the electric field of light as static, the polarizability can be calculated
easily for a sphere:
This is valid for particles that are small compared to the wavelength of light (<50 nm).
450 500 550 600 650 700 750 800 8500
1
2
3
4
5
6
7
8x 10
-15
cro
ss s
ecti
on
(σ
ext,
m2)
wavelength (λ0, nm)
25
20
15
10
5
R (nm)
AuH2O
geometric cross section areas
m
m0
23
εε
εεVα
Jackson
Classical Electrodynamics Wiley 1999
The symbol ε represents (relative)
permittivity at optical frequencies.
For simple transparent materials
like water, ε is roughly the square
of the refractive index (ε = n2).
For metals, the permittivity is
complex (energy absorption) and
dispersive (λ dependent).
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The Optical Near-Field
The absorption and scattering cross sections correspond to the “far field” properties of
the nanoparticles, i.e. they describe what happens to a light wave that passed the particle.
One can also talk about the “near-field”, i.e. the local electromagnetic field distribution
on the nanoscale. This is very important when using the particles as biosensors!
The field is strongest at the metal and
typically extends a distance approximately
equal to the radius of the particle.
Electrostatic theory can be used to
calculate the near field as well:
E0
(y, n
m)
z = 0
λ0 = 526 nm
(x, nm)
field enhancement (|E|/E0)
Au
H2O
zyx3x
Rex
y,,
5300
zyxr
x
rE
zxE
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Surface Plasmons
Surface plasmons are similar to ordinary light (electromagnetic plane wave) but confined
to the interface between a metal (conductor) and a dielectric (insulator).
The surface plasmon propagates along the interface (like a wave on a water surface).
Eventually the wave energy has dissipated (normally by heating the metal).
The wave must have transverse magnetic polarization.
x
y
z E field vector in z direction
H field vector in y direction
wave propagation in x direction
xiexpiωexp, 0
kxtEtxE yωiexp,, zx0
tzkxkHtzxH
metal
dielectricz
x
Re(kx)Im(kz)
H(x,z,t)
+ ++ + - -- - + ++ + - -- -
λSP
0
distribution
of E field
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The Dispersion Relation
There exists a relation between the momentum and energy of a surface plasmon. In
contrast to nanoparticles, surface plasmons exist in a continuum of frequencies!
Problem: Momentum is always lacking for the incident light! The dispersion relation
does not cross that of photons in the dielectric medium!
0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6
x 107
1.6
1.8
2
2.2
2.4
2.6
2.8
3
x 1015
real part of wavevector (Re(kx), m-1)
an
gu
lar
freq
uen
cy
(ω
, H
z)
photon in
vacuum
photon in
water
Au
H2O
m
mx
ck
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Excitation of Surface Plasmons
The additional momentum needed can be added in
several ways.
One is to introduce a periodic grating on the
surface. The grating wavevector adds to that of the
incident photons. The condition for excitation is:
One can also use a high refractive index material,
a thin metal film and total internal reflection
configuration. The resonance condition is then:
metal
dielectric Λ Re(kx)
θ
ksin(θ)
k
Re(kx)
metal
dielectric
prism
k0npsin(θ)
θ
k0
Λ
jn 0
m
m0
m0 sinRe
sinRe p
m
m n
analyze
reflection
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Total Internal Reflection
When measuring the reflected light one sees a minimum, representing SPR! One can vary
either the angle or the wavelength of incident light (spectroscopy at fixed angle).
0 10 20 30 40 50 60 70 80 900
0.2
0.4
0.6
0.8
1
angle of incidence (θ, )
Fre
snel
co
effi
cien
t
FT
FR
FAno full
reflection
resonance
Au d = 50 nm
H2O
np = 1.7
λ0 ≈ 630 nm
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
-2
-1
0
1
2
Ez
position in propagation direction (x, μm)
ele
ctr
ic f
ield
com
ponent (a
.u.)
Ex
λSP/4 Au
H2O
kx = 1.4 107 + 9.7 104i m-1 kz = -2.4 105 + 5.7 106i m-1 z = 0
2017-01-25 Biotechnical Physics 20
Surface Plasmon Near-Field
At any time, the surface plasmon has
negative and positive poles on the metal
surface.
The electric field has two components in
the xz plane. Magnetic field only in y.
The time-averaged field only depends on
the distance from the surface due to
symmetry.
The field extends approximately half of
the wavelength of light used to excite the
surface plasmon.
metal
dielectricz
x
Re(kx)Im(kz)
H(x,z,t)
+ ++ + - -- - + ++ + - -- -
λSP
0
distribution
of E field
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Checklist
You do not have to learn any equations for calculating stuff related to the optics, such as
how to predict the resonance of a certain nanoparticle etc. However, you need to
understand the basic physics.
Checklist of some important concepts and the differences between nanoparticle and
surface plasmons:
• Electrostatic approximation.
• Extinction, absorption and scattering cross sections.
• Dispersion relation and momentum of incident light.
• How to excite plasmons by light and spectroscopy methodology.
• Electromagnetic field extension, difference between near-field and far-field.
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Biosensing with Plasmonics
The most common plasmonic biosensor principle is refractometric detection:
• When a molecule binds to the surface, the refractive index changes. All molecules of
interest have a refractive index which is higher than water.
• The properties of the plasmon are changed because they depend on the refractive index
close to the metal.
• By optical spectroscopy, changes in intensity of light for different wavelengths can then
be detected. The resonance shifts in the spectrum.
This holds both for surface plasmons (the SPR technique) and nanoparticle plasmons (lab
exercise 2).
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Commercial SPR
• First paper published in 1983.
• Pharmacia Biosensor started shortly after. Became
Biacore later, which is now part of GE Heathcare.
• SPR is now the most established biosensor
technology for studying biomolecular interactions.
• But not used for medical diagnostics...
• The instruments are expensive, but primarily because
they contain efficient liquid handling and temperature
stabilization etc. SPR can be cheap!
Liedberg et al.
Sensors and Actuators 1983
2017-01-25 Biotechnical Physics 24
SPR Imaging
SPR can be operated in imaging mode which enables multiplexing: Several interactions
can be probed simultaneously using the same sample solution.
One molecule in solution: See how it interacts with different receptors on different spots.
Ideal for proteomics and in principle for drug development.
Several molecules in solution: Detect the presence of multiple analytes in one sample
using different recognition elements. High risk of problems from nonspecific binding.
~100 μm spots, ~100 in totalHomola
Chemical Society Reviews 2008
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Other Optical Techniques
Plasmons is definitely not the only way to go!
• Ellipsometry (often for air environment).
• Optical waveguide lightmode spectroscopy.
• Dual polarization interferometry.
Very similar since all are based on refractometric detection!
Microvacuum
http:/www.owls-sensors.com/
2017-01-25 Biotechnical Physics 26
Importance of Field Extension
If the electromagnetic field extends very little, molecules far away will not be detected.
If the field extends far, only a part of the detection capability is utilized and the system
becomes more sensitive to “bulk effects” far away from the surface.
nanoparticlehighest signalgel matrixmonolayer
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Miniaturized Nanoparticle Sensors
If SPR is so great, why bother with plasmonic biosensors based on nanoparticles?
One reason is that people want to resolve individual molecules binding to the surface.
SPR is hard to make sufficiently small, but through measurements on single
nanoparticles one can perhaps reach single molecule resolution?
Murray & Barnes
Advanced Materials 2008
scattered light in dark
field illumination
2017-01-25 Biotechnical Physics 28
Resolving Single Molecules
Very large proteins adsorbing directly on the surface.
Help of complementary techniques can make it slightly better…
Cool, but is it useful?
Ament et al.
Nano Letters 2012
Zijlstra et al.
Nature Nanotechnology 2012
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Sandwich Assays
Use secondary receptor and signal amplification post binding.
Improves detection limit, but excludes real time analysis to get kon and koff!
Ysecond tagged
receptor
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Competitive Assays
Measure the reduction in receptor binding to
the surface, which contains a receptor for the
receptor.
Target binding to the receptor in solution
blocks binding site for receptor on surface.
Excellent for small molecules and
interaction occurs in solution!
But again no kon and koff!Y Y Y Y
receptor in
solutiontarget
receptor on
surface
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Detection Based on Particle Coupling
Instead of detecting changes in
refractive index on the metal
surface, one can utilize the fact
that nanoparticles close to each
other will change their plasmon
resonances.
In general, the spectral changes
are much larger and the
sensitivity is better.
However, this requires that the
analyte either cleaves the link
between particles or couples
them together.
linked particles
free particles
sequence
recognition
by analyte
Y
Y
Y
Y
sandwich double
recognition
2017-01-25 Biotechnical Physics 32
Other Uses of Nanoparticles
Gold and silver nanoparticles have been used throughout history for “improving health”.
Also, they are great labels since they do not bleach!
Wikipedia: Argyria
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Reflections & Questions
?