Optical Sensors

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Optical Sensors

• Fluorescent Biosensors
• Total Internal Reflection
• Surface Plasmon Resonance
• Interferometry

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Fluorescent biosensor design
The fundamental design of a fluorescent biosensor
consists of a receptor binding site and a
fluorophore connected by a linker. The
linker provides a means for triggering a change
in the fluorescence of the attached fluorophore.
Fluorophore
Receptor/ Binder
Linker
Bissel et al. Topics in Current Chemistry, Vol.
168 , Springer Verlag (1993), pp. 223-261
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Fluorescent biosensors
The principal of operation is that there must be
a fluorescent switch. The switch is triggered by
a binding event. The binding molecule can
quench fluorescence or cause a conformational
change that unquenches fluorescence. One of the
most common quenching mechanisms is electron
transfer hn
DA D1A DA- DA
hn DA In the scheme
above the donor D acts as a fluorescence
quencher.
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Energy diagram for a fluorescent sensor
D 1A
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Electron transfer quenching of a fluorescent
sensor
Es
D A-
D 1A
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Fluorescent sensors
Crown ethers bind sodium with good
selectivity. In the fluorescent molecule shown
below the binding of sodium results in
fluorescence quenching.
Cooper and James SPIE (1999), 3602, 194-201
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Boronic ester-based biosensorsThe classical
mechanism
Formation of a boron-nitrogen bond can occur by
formation of boronic esters. This interaction
dramatically reduces fluorescence quenching by
the amine nitrogen lone pair. The result is a
switch from a non-fluorescent to fluorescent
state upon binding to a saccharide.
Cooper and James SPIE (1999), 3602, 194-201
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Novel mechanism for fluorescence triggering




Hydrolysis Less Favored
Hydrolysis Strongly Favored
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In aprotic solvents (DMSO, CH3CN, CHCl3),
fluorescence of the ester is lower than that of
the acid



Fluorescence profile of acid in anhydrous DMSO
decreased with the addition of cis-1,
2-cyclopentane diol
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Addition of water increase the fluorescence
intensity



DMSO and Water
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A Hydrolysis Mechanism for Fluorescent State
Switching




Franzen, Ni and Wang J. Phys. Chem. 2003, 107,
12942
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Implantable glucose sensor
Fluorescent molecules
Laser diode
SMSI, Inc.
Detector
Power supply and transmitter
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Implantable glucose sensor
Fluorescent molecules
Laser diode
SMSI, Inc.
Detector
Power supply and transmitter
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Implantable glucose sensor
Glucose quenches fluorescence
Laser diode
SMSI, Inc.
Detector
Power supply and transmitter
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Cell Surface Target Molecules




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The Approach




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Bidentate design



Computer-aided design can be used to generate
structures that have the appropriate geometry. A
recent example is shown above for sensing of
pyranose.
He and Druekhammer, Angew. Chem. 2001, 40, 1714
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Some Synthesized Diboronic Acids



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Fluorescent Cell Targeting Assay
Sialyl Lewis X Sialyl Lewis Y
Control
12q 12a
5 mM Boronic Acid Targeting Molecule
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Plasma Frequency
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The polarization model The free electron optical
response uses the Drude-Lorentz-Sommerfeld
model. The influence of external forces is
considered for one electron alone and then the
response is multiplied by the number of
electrons. All electrons act in phase in this
model. We consider an electric field
perturbation without local field corrections.
The response of a free electron of mass me and
charge e to an external electric field E
E0e-iwt is described by the drift motion
superimposed on the motion in the field-free
case Where G denotes a phenomenological
damping constant. This equation is readily
solved to give the dipole moment p er0 and
polarization P np ner0, where n is the
number of electrons per unit volume.
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Solution using the dielectric susceptibility Cons
ider isotropic media where PE and therefore P
c(w)e0E where c(w) is the (frequency dependent)
dielectric susceptibility and e0 is the
permittivity of vacuum. The polarization P
connects to the dielectric function e(w) by the
definition c(w) e(w) - 1 so that P (e(w) -
1)e0E. Thus, we can obtain an expression for the
equation of motion in terms of the
polarization and the perturbing electric
field where the frequency-dependence of the
response is contained in the dielectric
susceptibility.
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Solution using the dielectric susceptibility
(contd) We obtain a solution by substituting in
E E0e-iwt to obtain We cancel E0e-iwt on
both sides and solve for c(w). which can
also be written as
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The complex dielectric constant according to
Drude From the definition c(w) e(w) - 1 and
the definition of the Drude plasmon resonance
frequency we have The relaxation constant G
can be related to the electron mean free path l
and the Fermi velocity vF by G vF/l
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Nanoparticle Plasmon Resonance
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Plasmon Resonance Absorption
The plasmon resonance frequency wp is predicted
by the Drude free electron model. For a bulk
material
n is the number of free carriers e is the charge
on the electron
30 nm particle
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Home Pregnancy TestColloidal gold coated with
HcG antibody
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Home Pregnancy TestAnalyte is present
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Home Pregnancy TestGold aggregates
Absorption band shifts due to aggregation
and color changes from pink to blue.
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Surface plasmon resonance
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Surface plasmon resonance
Surface plasmon resonance (SPR) is a phenomenon
which occurs when light is reflected off thin
metal films. A fraction of the light energy
incident at a sharply defined angle can interact
with the delocalised electrons in the metal film
(plasmon) thus reducing the reflected light
intensity. The precise angle of incidence at
which this occurs is determined by a number of
factors. In biosensor applications the principal
determinant becomes the refractive index close to
the backside of the metal film. Target molecules
are immobilised and addressed by ligands. If
binding occurs to the immobilised target the
local refractive index changes, leading to a
change in SPR angle, which can be monitored in
real-time by detecting changes in the intensity
of the reflected light. These changes can be used
to determine affinity constants.
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SPR implementation
Reflected Light
Incident Light
I
Flow Cell
Molecules in solution exhibit changes in
refractive index and give rise to a measurable
SPR signal when binding occurs.
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SPR implementation
Incident Light
Reflected Light
II
Flow Cell
Molecules in solution exhibit changes in
refractive index and give rise to a measurable
SPR signal when binding occurs.
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SPR applications

• Ligand - Receptor interaction
• Antigen-Antibody interaction
• Protein - Protein interaction
• RNA - Protein interaction
• DNA - DNA interaction
• Cell - Protein interaction
• Drug Screening
• Real-time monitoring for gene expression

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We can consider the general case of light
impinging on a surface. There are three rays we
can follow i incident r reflected t
transmitted or refracted There are two processes
that are considered in basic physics courses.
Reflection qr qi Refraction nrsinqr
nisinqi (Snells law) For plasmons p-polarized
light must be used. The electric vector is
parallel to (in) the plane of incidence.
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n2 lt n1
z
kr
ki
qi
qr
n1
x
y
n2
qt
kt
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Maxwells equation leads to wave equation
The solution is a plane wave
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The dispersion is obtained by plugging this
solution into the wave equation Dispersion
refers to a frequency dependence. There is a
dispersion relation for molecular absorption
(i.e. it depends on frequency). Here the
dispersion is the dependence of the wavevector k
on frequency. Recall that the wavevector
is related to the spatial component of the
radiation. It is the angle of incidence that
changes the spatial component of surface
incidence.
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z
Red double arrow gives the electric vector of
the incident radiation.
x
y
ki
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z
x
y
ki
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z
Note how the projection of the wave on the
surface changes with angle.
x
y
ki
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Now we include the fact that there are two
different indices of refraction on either side of
the surface. Stokes theorem states that the
tangential E and H must be continuous. The unit
vector y is the direction Of the magnetic vector.
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How can the different k values match up?
z
ki
kr
x
y
kt
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Fresnel Equations
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Fresnel Equations To calculate the relative
intensities of reflected and refracted waves in
terms of the energy of the incident wave, we
introduce Fresnel's equations. We consider the
two polarizations of electromagnetic radiation
at the interface between two media of different
indices of refraction (n1 ¹ n2). The derivation
here applies for non-magnetic and non-conducting
media (the magnetic permeability m1 m2).
s-polarized
p-polarized
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• We first consider the case where light propagates
  in medium 1
• with index of refraction n1 and impinges medium
  with n2 gt n1.
• This situation is common in our experience since
  light often
• Propagates in air and then reflects off of water
  or glass. The
• issues are
• What is the probability of reflection of the
  light as a function
• of the angle incidence? (Fresnel equations)
• What is the angle of transmission? (Snells law)
• What is the phase change of light for reflection
  and
• transmission?
• 4. What is Brewsters angle?

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s-polarized, n1 lt n2
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s-polarized, n1 lt n2
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s-polarized, n1 lt n2
Using the proportionality between the wavevector
and the index of refraction
In vacuum
In a medium
Since the frequency does not change as radiation
crosses the boundary and c is the speed of light
k is proportional to n.
50
p-polarized, n1 lt n2
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p-polarized, n1 lt n2
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p-polarized, n1 lt n2
s-polarized
p-polarized
53
Intensity of reflectance for p- and s-polarized
light
n1 lt n2
Brewsters angle
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Phase change upon reflection
s-polarized does not change phase if n1 gt n2
s-polarized changes by p if n1 lt n2 (Et changes
sign) For p polarized light, the reflected beam
the ratio Er/Ei can be either negative or
positive depending on both the ratio n1/n2 and
the incident angle qi. The Er component is in
phase with Ei at the interface if Using
Snells law and the sum and difference formulae
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Phase change upon reflection
The equation will be satisfied if Thus under
these conditions the reflected p-polarized
beam will not undergo a phase change.
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The Brewster angle
The electric field intensity of the reflected
wave is either in-phase or p radians
out-of-phase with the incident wave, depending on
whether sin(qt - qi)cos(qt qi) is greater than
or less than zero. There is no reflected wave
when sin(qt - qi)cos(qt qi) 0, that is when
qi qt 0 or qi qt p/2. The latter
condition is called Brewsters angle. Note that,
since all p-polarized light is refracted, any
light reflected from the interface at this angle
must be s-polarized. A glass plate or a stack of
plates placed at Brewster's angle in a light beam
can thus be used as a polarizer. For a glass
medium (n21.5) in air (n11), Brewster's angle
for visible light is approximately 56 to the
normal while for an air-water interface
(n21.33), it's approximately 53. Since the
refractive index for a given medium changes
depending on the wavelength of light, Brewster's
angle will also vary with wavelength.
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Total internal reflection

• Only occurs when n1 gt n2
• The critical angle
• Key geometries
• Conductors vs. Insulators

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Total Internal Reflection
If the angle of incidence is greater than the
critical angle, the wave originating in medium 1
and incident on the interface of two media is
totally reflected back into medium 1. This
phenomenon which is called total reflection,
does not depend on the polarization of the E
vector in the incident wave. It turns out that
Snell's law and Fresnel's equations are
applicable to total reflection if disregard the
fact that sinqt gt 1. .
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Total Internal Reflection
If we now return to the coordinate system used
for the study of reflection and refraction we
can write the waves as The reflected
wave is exactly the same for total reflection as
for any other reflection.
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Total Internal Reflection
The transmitted wave can be expressed in terms of
the condition for total reflection and the above
expression for cosqt. The transmitted wave
(in medium 2) has negative values of z.
Therefore, the wave consists of a traveling
component in the x direction and a decaying
wave z-direction
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Attenuated total reflection angles q gt qc
Ep Ei
Es Ei
p polarization s polarization
rp 1
ea


rS 1
-
-
z
e0
x
Evanescent Wave in Medium
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The dispersion relation

• Propogation in high index medium
• Total internal reflection
• Wavevector matching condition
• Parallel and perpendicular components

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The electric field at the interface
The coupled fields inside (-) and outside () the
solids are given by where The electric field
of the surface plasmon (SP) represents a
harmonic wave along the surface with a frequency
w and a wave vector kx, while perpendicular to
the surface (z-direction) the fields are
exponentially decaying. The boundary conditions
require that
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The SPR Dispersion Relation
es is the dielectric constant of the metal
(substrate) ea is the dielectric constant of the
sample For the metal the dielectric constant
is complex and can be derived from the index of
refraction.
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Optical constants for ITO
ITO is chosen as an example of a conducting thin
film
Complex index of refraction
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Dielectric constants for ITO
e1 e2
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ITO Dispersion Curve
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Light will couple to drive a surface plasmon
where the light line crosses the dispersion curve.
Light lines
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ITO Dispersion Curve (expanded)
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SPR uses a three layer system Attenuated total
reflection (qi gt qc )
p polarization s polarization
eIRE


-
-
em
es
Evanescent Wave in Medium
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Kretschmann configuration
eIRE gt
es


p polarization s polarization
-
-
eIRE
em
es
Thin film on a prism
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Plasmon bands for different indices of refraction
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Interferometric Sensors
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Biosensor for rapid detection of microbes
Based on interaction of light with binding of
bacteria. The interference in two beams can be
detected.
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Integrated optical interferometric sensor
Reference
Sample
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Integrated optical interferometric sensor
Reference
Sample
Pathogenic Bacteria
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Integrated optical interferometric sensor
Reference
Sample
Binding to Substrate
Phase shift of light
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Interferometer

Constructive addition Two light waves with the
same phase sum to give a greater amplitude
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Interferometer

Cancellation Two light waves that are 180o out
of phase sum to give zero amplitude