Modern methods of measuring thin film formation and kinetic analysis Dr' Toby Jenkins

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Modern methods of measuring thin film formation
and kinetic analysisDr. Toby Jenkins

• CH20016
• Lectures 9 and 10
• http//staff.bath.ac.uk/chsataj

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Synopsis of 7 Lectures

• Lecture 1 Measurement of molecules adsorbing on
  surfaces.
• Lecture 2 Kinetic analysis of equilibrium and
  dynamic measurements.
• Lecture 3 Surface IR and Raman.
• Lecture 4 Scanning Tunnelling and Force
  Microscopies.
• Lecture 5 Introduction to electrochemistry.
• Lecture 6 Mass transport in electrochemistry
• Lecture 7 Electrochemical methods Cyclic
  voltammetries

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The measurement and analysis of molecules,
proteins etc. with structured surfaces

• Why are interested in measuring this?
• Methodology for obtaining association constants
  between a surface bound receptor e.g. a protein
  and an analyte e.g. an antibody.
• Important for construction of biosensors sensors
  that measure biological analytes.
• Important in molecular and structural biological
  analysis
• of proteins.

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Example of a biosensor Home pregnancy test kit

• Commercial pregnancy test kits are remarkably
  sensitive detect down to nM (10-9) or lower
  concentrations of human chorionic gonadotrophin
  (hCG).
• Human chorionic gonadotrophin is an enzyme which
  increases in concentration geometrically post
  conception, during foetal growth.
• Detected in urine
• Monoclonal antibodies which only bind this enzyme
  have been developed anti-a-hCG, binds to a
  domain and anti-b-hCG binds to b domain on hCG.

?
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hCG detection by antibodies
Fab specific protein recognition
Fc end
Antibody structure
hCG
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The ELISA (Enzyme Linked Immunosorbent Assay)
Commercial hCG sensor uses a hydrophilic wicking
strip, when labelled reporter antibodies
associate on reporter strip, see blue or pink
colour due to density of labels attached to
reporter antibody
non-bound, reporter anti-b-hCG
bound catcher, anti-a-hCG
Sample (urine) here
Wicking direction
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Determination of antibody proteinbinding
constants

• In designing a biosensor system it is important
  to be able to measure rate of association kass
  between a protein and antibody and rate of
  disassociation kdiss.
• Consider a surface bound antibody A and a
  solution phase protein P

Equilibrium constant KA and KD for this system
Rate of forward reaction kassAP, rate of
back reaction kdissAP
So, if we can measure association and
disassociation rate constants directly, get
equilibrium constant
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Strategies for measuring association of molecules
/ proteins with surface bound receptors

• Brief Overview
• Quartz Crystal Microbalance A resonating quartz
  crystal changes its frequency of resonance as
  molecules bind on top a very sensitive mass
  balance.
• Surface Plasmon Resonance Interaction of light
  with a thin metal film creates an energy wave.
  Angle at which this resonance takes place is
  related to the dielectric constant of material
  adsorbing on surface.
• Fluorescence assays measure increases in
  fluorescence as fluorescently labelled proteins
  or molecules bind to surface.

All systems give a response which is proportional
to the concentration of bound analyte
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The Quartz Crystal Microbalance
Measure mass change on binding of an analyte to
an oscillating quartz crystal
The resonant frequency (f) of the crystal depends
on the total oscillating mass, including water
coupled to the oscillation. When a thin film is
attached to the sensor crystal the frequency
decreases. If the film is thin and rigid the
decrease in frequency is proportional to the
mass of the film. In this way, the QCM operates
as a very sensitive balance. The mass of the
adhering layer is calculated by using the
Sauerbrey relation C 17.7 ng Hz -1 cm-2 for
a 5 MHz quartz crystal. n 1,3,5,7 is the
overtone number. It is also possible to get an
estimation of the thickness (d) of the adhering
layer,  where reff  is the effective density of
the adhering layer.
Check out www.q-sense.com
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Example QCM measuring bacterial attachment to
surfaces
1. Initial attachment of bacteria 2. Growth and
division on surface. 3. Proposed movement away
from surface in slime mattrix
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DD (on RHS) is the dissipation factor, which
relates to visco-elasticity of adsorbed material
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Surface Plasmon Resonance
Introduce P polarised light into a thin gold
film. Measure reflected light intensity as a
function of incident light angle At a certain
angle, light is not reflected but interacts with
free electrons in gold to form a resonant energy
wave (surface plasmon). Angle of incidence
sensitive to changes in refractive index of
dielectric. Hence, measure real time changes in
dielectric as a thin film / molecules / proteins
bind to surface.
Check out http//home.hccnet.nl/ja.marquart/
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SPR information obtained
The reflectivity time curve can then be used
for kinetic analysis
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Colourimetric / light absorbance / fluorescence
assays

• Usually run in a microtitre plate format for
  statistical validation of results and parallel
  screening possibilities. Label secondary
  antibodies (for example) with
• a reporter.

• Measure directly either
• Light absorption at a wavelength
• Ae/cl
• Fluorescence emission
• Colour

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Rate of formation of surface bound
protein-antibody complex

So, we measure a response, R proportional to rate
of formation of surface bound AP complex
Assume Langmuir isotherm

• Clearly, the number of free site A will
  decrease with time, so we can write
• Where APt is the concentration of bound sites
  at time t.
• So substituting

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Simplifying
Using a response from an analytical measurement
APt ? Rt Moreover, maximum response Rmax is
equivalent to Ao since all binding sites
are occupied (Langmuir assumption) so we can
write
This equation is very important as it relates the
time dependent measured response to the kinetic
parameters kass and kdiss
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Towards equilibrium, dRt/dt ? 0


The gradient of this curve at a time t, dRt/dt is
given by
Typical binding isotherm
At equilibrium


The Langmuir equation!

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Langmuir equation
Rmax
(here get 4 points on RHS graph)
Kinetic measurements at different concentrations
of P
At high concentrations of P and / or low of
kdiss, Req ? Rmax
When Req Rmax / 2 P KD So KD is conc. of
protein to cover half the surface sites.
So we have derived the Langmuir equation, in
terms of a measured response from any system
which gives a signal proportional to the
concentration of the protein-antibody complex
and shown how we can determine KD directly.
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Dynamic kinetic analysis determination of
kinetic constants and KD for systems which dont
come to equilibrium
Solve this differential equation
but remember
where kon kassP kdiss
kon is a pseudo 1st order rate constant
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Using real time data

• Obtain minimum three Response vs time curves
  (for three analyte concentrations)
• Fit data to equation
• Obtain kon for each concentration.
• Plot Kon vs. analyte to get a linear plot with
  gradient kass and intercept kdiss.

Pale blue lines are fit, data relates to binding
of anti-BSA to a BSA modified surface
kass 5178 /- 1623 mol-1 dm3 s-1 and kdiss
0.0028 /- 0.00074 s-1 giving value of KD 5.48
x 10-7 mol dm-3.
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Direct determination of kdiss

• So far have considered a situation where there is
  a constant concentration of binding analyte in
  bulk solution.
• Consider situation where surface is rinsed with
  buffer, such that all analyte in bulk solution is
  removed Pt0.

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So can determine kdiss by plotting ln(Ro/Rt) vs.
t, giving gradient of kdiss
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Summary

• We have seen how measuring any response, which
  gives a signal proportional to the concentration
  of surface bound analyte APt, we can determine
  rate of association (kass), rate of
  disassociation (kdiss), and thermodynamic
  equilibrium constants, KA and KD for surface
  bound system.
• We have explored two methods to obtain KD, from
  equilibrium measurements, using the Langmuir
  equation directly, and from non-equilibrium
  measurements, where APt is measured as a
  function of time.
• Finally, we saw how kdiss can be measured
  directly in an experiment, by measuring a
  response during rinsing of the surface.
• Dont think about becoming pregnant until you
  understand all this!