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K. Castelino 2/19/2004
Protein Dynamics and Structure using Broadband Dielectric Spectroscopy
Kenneth Castelino, Veljko Milanović, Dan McCormick
Nanoengineering Laboratory
Dept. of Mechanical Engineering
University of California at Berkeley
K. Castelino 2/19/2004
• Motivation and Introduction to Proteins
• Dipole Relaxation Phenomena (kHz - GHz)
• Terahertz Spectroscopy
• Conclusions
Outline
K. Castelino 2/19/2004
Motivation
Borrowed from:Department of BiostatisticsHarvard School of Public HealthJanuary 23-25, 2002Sandrine Dudoit and Robert Gentleman
K. Castelino 2/19/2004
Protein Structure
Lehniger Principles of Biochemistry, Worth Publishers, NY, 2000
1.
• Peptide Bonds• 20 Amino Acids
2. Secondary Structure
-helix -sheet
• Hydrogen Bonds
3.
• Noncovalent Interactions
K. Castelino 2/19/2004
Protein Structure
Lysozyme (43% , 5% , 52% r.c.)
-lactoglobulin (6% , 45% , 49% r.c.)
-lactalbumin (33% , 17% , 50% r.c.)
Pepsin (13% , 59% , 28% r.c.)
K. Castelino 2/19/2004
• Broadband spectral “signature” of a protein• Specific complex permittivity of the solution
• Protein structure• Identify spectral features that provide info about structure.• “Invert” the spectral signature to learn more about the
biomolecule
• Protein Function• Changes in relaxation or resonances due to binding. • Changes in long range vibrational modes.
What are we looking for?
K. Castelino 2/19/2004
Frequency Range
Relaxation Phenomena – Debye Dipoles
Resonant Phenomena – Lorentz Oscillators
K. Castelino 2/19/2004
Interaction of Matter and Radiation
)(2
2
teEkxdt
dxf
dt
xdm =++
m
k
f
Eejwt
Lorentz Model
K. Castelino 2/19/2004
Dispersion of dielectric constant
Dielectric constant of an aqueous hemoglobin solution
Dielectric constant
Radio wave Microwave IR Visible UVn
Refractive index
n
n
Absorption
Dipole Relaxation
Resonance
K. Castelino 2/19/2004
Low Frequency - Relaxations
• LF => 1 kHz to 100 MHz• Surface binding detection – change in
capacitance due to charges or displacement of charges or dielectrics
• In solution (essentially ‘re-arranged water’)
+ -
++ + + +
• HF => 100 MHz to 110 GHz• Same as above possible• Also see dielectric relaxations of solvent ions• Possible resonances due to bio-molecular structure
(secondary, tertiary).• Proteins as ‘box of springs’ (or re-arranged water)
K. Castelino 2/19/2004
Measurement Setup
• Agilent / Anritsu PNA
• Frequency:• 45 MHz - 50GHz 601 points • Power Level: 5 dBm
• TRL / SOLT calibration. • To probe tips
Anritsu Corp.
K. Castelino 2/19/2004
Protein fingerprinting in RF field
Microstrip
h
w
Coplanar
w1
w2
ε r
Waveguide
Twisted-pair
Coaxial
b
a
h
Some standard transmission line types
Fluid sample
Microchannel/fluid cavity
CPW on nitride membrane
ME
_C
PW
3 c
hip
• Options of transmission line types – coax, waveguide, on-substrate
• Main challenge is broad frequency range and fluidic capability
K. Castelino 2/19/2004
CPWs for bio-testing – the main test device
• Al CPWs
• Nitride/oxide membrane
• For on-wafer probing network analyzer characterization
• Liquid held by surface forces
• Two holes for bubble release and easier fabrication
200 µm
back front
TESTdevice
K. Castelino 2/19/2004
LF measurements example: Lysozyme in 1mM acetate buffer solution
Sensitivity inversely proportional to ion conc. Due to double layer effects
K. Castelino 2/19/2004
LF measurements example: DNA immobilization and hybridization
Frequency [Hz]
1e+3 1e+4 1e+5 1e+6 1e+7
Capacitance [F]
5.0e-9
1.0e-8
1.5e-8
2.0e-8
2.5e-8
Immob. SS DNA Immob. SS DNA + mismatch DNA Immob. SS DNA + compl. DNA 15 min
K. Castelino 2/19/2004
CEAABSAPSABuffer
S2
1 [d
B]
10 Freq [GHz] 110
High Frequency Measurements
K. Castelino 2/19/2004
Liquid Nitrogen Experiments
S21
(d
B)
253 K
200 K
77 K
• Improve Q.
• Minimize thermal contributions.
K. Castelino 2/19/2004
LN2 Measurement Difficulties
K. Castelino 2/19/2004
Nanogap Transmission Lines
K. Castelino 2/19/2004
Transmission: Acetate Buffer vs. Lysozyme
K. Castelino 2/19/2004
Terahertz Experiments
• Frequency Range: 500 GHz – 2 THz.
• Amplitude + phase of E-field measured Get (ε’, ε’’) simultaneously
• Sensitive to sample preparation, thickness.
K. Castelino 2/19/2004
Terahertz Generation & Detection
Pulse Detection
Pulse Generation
K. Castelino 2/19/2004
Conclusions
• Only relaxations observed upto 100 GHz.
• Very sensistive binding changes observed in the double layer capacitance.
• Possible to see vibrational modes in the range of 0.5-3THz.
• Low temperature required in order to decipher modes with reasonable Q.
K. Castelino 2/19/2004
Acknowledgements
• Anritsu Corporation – Arno Pettai et al• Measurement facilities
• UC Davis – Prof. Norman Tien• Measurement facilities
• NIST Boulder – Dr. Dylan Williams• Assistance with calibration
• Lawrence Berkeley Lab – Prof. Dan Chemla• Terahertz Time-Domain Measurement.
K. Castelino 2/19/2004
TransmittedIncident
TRANSMISSION
Gain / Loss
S-ParametersS21, S12
GroupDelay
TransmissionCoefficient
Insertion
Phase
ReflectedIncident
REFLECTION
SWR
S-ParametersS11, S22
ReflectionCoefficient
Impedance, Admittance R+jX
, G+jB
Return
Loss
Incident
Reflected
TransmittedRB
A
A
R=
B
R=
High-frequency device characterization
Network Analyzer Basics
K. Castelino 2/19/2004
Measuring S-Parameters
S 11 = Reflected
Incident=
b1
a 1 a2 =0
S 21 =Transmitted
Incident=
b2
a 1 a2 =0
S 22 = Reflected
Incident=
b2
a 2 a1 =0
S 12 =Transmitted
Incident=
b1
a 2 a1 =0
Incident
TransmittedS
21
S11
Reflectedb1
a1
b2
Z0
Loada2 =0
DUTForward
Incident
Transmitted S
12
S22
Reflected
b2
a2b
a1=0
DUTZ0
Load Reverse
1
K. Castelino 2/19/2004
Systematic Measurement Errors
A B
SourceMismatch
LoadMismatch
Crosstalk
Directivity
DUT
Frequency response reflection tracking (A/R) transmission tracking
(B/R)
R
Six forward and six reverse error terms yields 12 error terms for two-port devices
K. Castelino 2/19/2004
Types of Error Correctionresponse (normalization)
simple to perform only corrects for tracking errors stores reference trace in memory,
then does data divided by memoryvector
requires more standards requires an analyzer that can measure
phase accounts for all major sources of systematic
error
S11 m
S11 a
SHORT
OPEN
LOAD
thru
thru
K. Castelino 2/19/2004
Microwave Properties of Ice
Fujita et al
Zhang et al, APL, 7/01
Matsuoka et al
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