4g2: biosensors lecture 3 - university of cambridge
TRANSCRIPT
Kinetics of immobilised biological systems
• In many biological systems, binding recognition events between a ligand and receptor occur in both solution and at interfaces (typically cell membranes). Examples include antibody-antigen type interaction (solution) or the biological recognition involved in the opening of channels in membranes (interfaces)
Consideration – solution versus solid interface
• Solution– Isotropic distribution of binding partners.– Homogenous microenvironments for binding
sites.
• Solid interfaces– Spatial separation of bound compounds from
free compounds.– Employ surface sensitive techniques to
measure bound complex.
Considerations – solid interfaces
• The high local concentration of binding partners at the surface may result in large local concentration gradients of the binding partner in solutioninfluencing this process.
• Binding may not be independent of the environment and neighbouring sites.
• Orientation, alignment and accessibility of the binding site of the immobilised species becomes important.
• How do you practically engineer a functional binding surface such that it is homogenous and each binding site is equivalent?
• Need to suppress non-specific binding on the surface.
Langmuir Model
• Models steady state response of the sensor modulated by binding events on surface.
• Models an equilibrium process of binding between ligandand receptor under the following assumptions– Binding sites are independent of each other and freely
accessible to the binding partner in solution.– No non-specific binding on surface or interaction between
ligands.– Concentration of molecules on surface is low enough that it does
not substantially deplete molecules in solution and hence lead to concentration gradients.
– There is no transport limitation. In other words, an infinitely fast diffusion from solution to surface is assumed.
Mass transport
• In reality, the concentration of reactants and products can change locally at the sensor surface, resulting in a local concentration gradient which in turn may affect the reaction kinetics.
• Mechanisms affecting mass transport include:– Diffusion– Convection– Directed migration (e.g. movement of charged
species under a potential gradient)
Outline
• Acoustic Transducers– Bulk Acoustic Waves– Surface Acoustic Waves– Surface generated bulk acoustic waves
• Bulk Acoustic Wave Sensors– Quartz Crystal Microbalance
• Surface Acoustic Wave Sensors• Studying biomolecular interactions using
acoustic wave sensors
Acoustic Wave Biosensors
G. Cote et al, IEEE Sensors Journal, 2003.
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Acoustic Waves
• Involves deformation of the solid material – individual atoms are set into motion– interatomic forces change– internal restoring forces return material to equilibrium
• Deformation is time-variant– motion of the individual atoms are set by a balance of
restoring forces and inertial effects– each atom oscillates about its equilibrium position
• If the material is elastic � propagating wave is elastic or acoustic
• At resonance, the wave propagates through the solid medium with minimum energy loss
Spectrum of Acoustic Waves
Bulk Acoustic Wave
• Acoustic Waves that propagate through the bulk of the solid medium
• Longitudinal Waves– Atoms vibrate in the direction of wave propagation
• Shear Waves– Atoms vibrate in the plane normal to the wave
propagation• In an infinite solid elastic medium, for a given
direction of propagation, a longitudinal bulk wave and two degenerate shear waves can propagate.
Surface Acoustic Waves
• Two-dimensional wave propagation on the surface of elastic media
• Example: Acoustic energy released by an earthquake propagating on the earth’s crust.
• Amplitude of the wave decays exponentially as a function of depth from the surface.
• Typically, the amplitude is negligible a few wavelengths below the surface of the elastic solid
Surface generated BAW
• Surface Skimming Bulk Wave (SSBW): particle displacement is parallel to surface and normal to direction of propagation
• Acoustic Plate Mode (APM) or Reflected Plate Wave (RBW): Shear horizontal waves that propagate in the bulk of a piezoelectric medium. Piezoelectric plates act as acoustic waveguides confining the energy in the bulk of the plate as the wave propagates through multiple reflections of the top and bottom surfaces.
Acoustic Waves• TSM - Thickness Shear Mode, a
bulk acoustic wave travelling through the thickness. This is the basis for the Quartz Crystal Microbalance.
• SAW – Surface Acoustic Wave, Surface confined acoustic waves that propagate both longitudinal and transverse directions.
• APM – Acoustic Plate Mode, Surface generated acoustic waves that travel along the length and thickness of the device. The surfaces act as waveguides for the travelling wave.
Quartz Crystal Microbalance
Piezoelectricity• Utilise piezoelectricity for the excitation and detection of the waves• Piezoelectric materials exhibit an electrical potential between
deformed surfaces and the application of a voltage induces physical deformation in the material.
• Piezoelectric materials are crystalline anisotropic solids• Examples
– Quartz– Zinc oxide– Lithium niobate– Lead zirconate-titanate– Aluminum nitride– poly (vinylidene fluoride)
• Quartz is the most common choice for biosensor applications because of its chemical stability in aqueous solutions and relative resistance to high temperatures
• Quartz also benefits from considerable manufacturing know-how and the potential for micromachining
Surface Acoustic Wave Devices
Thickness Shear Mode resonators
• consists of parallel plate of crystallinequartz with electrodes on both sides• Fundamental mode wavelength is twice thickness of the plate.• Resonant frequency is given by:
- For a 0.33mm thickness, this translates to about 5MHz
dv
f2
=
Applications
• Thickness film monitoring• Gas sensors• Back-end detection for chromatography
columns• Biological applications
– Immunosensors– DNA hybridisation– Single celled organisms
Studying biomolecularinteractions
• First use for protein sensing in 1990s.• Operating principle: mass deposited on the
surface of the device will oscillate synchronously with the device surface under the influence of a propagating wave
• For biomolecular interactions, it is essential that these devices operate efficiently in liquid environments
Studying biomolecularinteractions
• Propagation characteristics of the wave are modulated by:– mass deposited on the device surface– changes in viscosity of the liquid– changes in electrical properties of the interface– roughness and wetability of the solid surface– nature of the interfacial layers
• Typically use a reference device to minimize interference in acoustic wave measurements
Some Theory
• Sauerbrey (1959) formulated a relation between the TSM resonant frequency and deposited mass on the surface of the crystal.
• Analytical expression is given by:
• Assumes that the frequency change is caused by mass loading only in vacuum.
• When a device is immersed in a liquid sample, energy is lost due to visco-elastic coupling.
µρµρµρµρAm
ff∆−=∆ 2
02
Sauerbrey Equation
fo – nominal resonant frequencyvtr – shear acoustic velocityd – crystal thicknessµq- shear modulusρq – density∆d – thickness of deposited layer∆f – shift in resonant frequency∆m – deposited massA – active sensor areaC – mass sensitivity
mf
C
Am
ff
Am
ff
dfdd
ff
ddv
f
qqo
q
q
qo
q
qo
o
q
qtro
dd
2
2
2
21
2
2
2
=
∆⋅−=∆
⋅∆⋅−=∆
∆⋅⋅−=∆−=∆
⋅==
µρ
µρ
ρ
µρ
ρµ
Effects of liquid viscosity
• For a pure viscous medium, the frequency shift is given by:
• In practice, there is a combination of mass loading and viscoelastic effects that result in a frequency shift.
llffµµµµπρπρπρπρ
ηηηηρρρρ2/30−=∆
Penetration Depth
Acoustic Wave Device
δ
solid/liquidinterface
ωωωωρρρρηηηηδδδδl
l2=
Equivalent Electrical Circuit
• It is possible to construct an equivalent electrical circuit for the quartz crystal resonator that captures frequency shift dependencies
• Often a parasitic capacitor, Co is added as a static feedthroughcapacitance between the two electrodes of the TSM resonator
• Resonator can be characterised open-loop or built into an oscillator.• This electrical model can be related to open-loop measurements and
characterisation data on electrical admittance and impedance.• The resonant frequency shifts can be measured using a number of
standard techniques including frequency counters.
Lq Cq Rq
Co
Loaded QCM
Martin et al, 1991
Admittance – 1st and 3rd modes
Martin et al, 1991
Admittance as a function of viscosity-density product
Martin et al, 1991
(a) Air, (b) Water, (c) 43% glycerol in water, (d) 64% glycerol in water, (e) 80% glycerolin water
Gold Deposition
Martin et al, 1991
A – air, B – water, C – air (with Au deposited), D – water (with Au deposited). Au isdeposited to a thickness of 124nm.
Dissipation Monitoring• Biomolecules and cells are often like jelly; they do not
respond as dead mass but are deformed in a manner that depends on their size, shape and viscoelasticproperties.
• Measure both amplitude and phase (frequency) or in other words both energy storage and dissipation.
• The damping is a parameter that can be extracted from the electrical response of the resonator.
• The physical models involved are typically much more complex.
• The resonant (or motional) resistance can be written as:
22/1 /)2( kAfR Lq ηρπ=
Resistance-Frequency Plots
• (a) – Elastic behaviour
• (b) – Viscous behaviour
• (c) – Variation with viscoelasticproperties of the coated thin film
• (d) – Summary of results
Muramatsu et al, 2002
Experimental verification – mass loading
Experimental verification –viscous loading
Water – Glycerol solutions are used to calibrate viscous loading effects on the crystal
Alcohol sensor
Muramatsu et al, 2002
Stepped injections of ethyl alcohol in gas phase and measured with a carbon film coatedQCM device. The sensor is close to linear in the range 1-120ppm.
DNA Hybridisation
T indicates binding complement and M indicates complement with one-base mismatch.The experiment is done for a number of DNA sequences of different lengths.
QCM – E. Coli detection
Spangler et al, 2001
Response of a QCM to increasing concentrations of a toxin produced by E.Coli withbinding specificity obtained by tailoring the surface with a protein binding receptor,ganglioside, GM1. This paper also included a comparison with an SPR systemdemonstrating a 2X increase in sensitivity.
STM protein
P. Ko Ferrigno et al
Surface density – CDK2
0 5000 10000 15000 20000 25000 30000
-50
-40
-30
-20
-10
0
10
Antibody -CDK2
Lysate-CDK2
PEGSTM-pep2
Freq
uenc
y S
hift(
Hz)
Time(s)
F:1 (Hz) F:2 (Hz) F:3 (Hz)
AFM imaging on mica surface Quartz Crystal Microbalance
Shu et al, 2007
Antibody-Antigen binding
0 5000 10000 15000 20000 25000 30000
-50
-40
-30
-20
-10
0
10
Antibody -CDK2
Lysate-CDK2
PEGSTM-pep2
Freq
uenc
y S
hift(
Hz)
Time(s)
F:1 (Hz) F:2 (Hz) F:3 (Hz)
Adsorption �f/n Mass Density Number Density Area/protein
STM molecule 5.0~5.03 Hz 88.5~89.0 (ng cm-2) 0.0487 Protein/nm2 20.5 nm2/protein
STM Antibody 3.0~3.2 Hz 53.1~56.6 (ng cm-2) 0.005protein/nm2 222 nm2/ antibody
Shu et al, 2007
Biotin-streptavidin binding
FLOW CELL CROSS-SECTION ADSORPTION ISOTHERM FOR BIOTIN
