K. Castelino 2/19/2004 Protein Dynamics and Structure using Broadband Dielectric Spectroscopy Kenneth Castelino, Veljko Milanović, Dan McCormick Nanoengineering

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


• Motivation and Introduction to Proteins

• Dipole Relaxation Phenomena (kHz - GHz)

• Terahertz Spectroscopy

• Conclusions

Outline


Motivation

Borrowed from:Department of BiostatisticsHarvard School of Public HealthJanuary 23-25, 2002Sandrine Dudoit and Robert Gentleman


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


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.)


• 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?


Frequency Range

Relaxation Phenomena – Debye Dipoles

Resonant Phenomena – Lorentz Oscillators


Interaction of Matter and Radiation

)(2

2

teEkxdt

dxf

dt

xdm =++

m

k

f

Eejwt

Lorentz Model


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


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)


Measurement Setup

• Agilent / Anritsu PNA

• Frequency:• 45 MHz - 50GHz 601 points • Power Level: 5 dBm

• TRL / SOLT calibration. • To probe tips

Anritsu Corp.


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


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


LF measurements example: Lysozyme in 1mM acetate buffer solution

Sensitivity inversely proportional to ion conc. Due to double layer effects


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


CEAABSAPSABuffer

S2

1 [d

B]

10 Freq [GHz] 110

High Frequency Measurements


Liquid Nitrogen Experiments

S21

(d

B)

253 K

200 K

77 K

• Improve Q.

• Minimize thermal contributions.


LN2 Measurement Difficulties


Nanogap Transmission Lines


Transmission: Acetate Buffer vs. Lysozyme


Terahertz Experiments

• Frequency Range: 500 GHz – 2 THz.

• Amplitude + phase of E-field measured Get (ε’, ε’’) simultaneously

• Sensitive to sample preparation, thickness.


Terahertz Generation & Detection

Pulse Detection

Pulse Generation


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.


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.


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


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


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


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


Microwave Properties of Ice

Fujita et al

Zhang et al, APL, 7/01

Matsuoka et al

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K. Castelino 2/19/2004 Protein Dynamics and Structure using Broadband Dielectric Spectroscopy Kenneth Castelino, Veljko Milanović, Dan McCormick Nanoengineering