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Introduction to BioMEMS & BionanotechnologyLecture 3

R. BashirR. Bashir

Laboratory of Integrated Biomedical Micro/Nanotechnology andApplications (LIBNA), Discovery Park

School of Electrical and Computer Engineering,

Weldon School of Biomedical Engineering,

Purdue University, West Lafayette, Indiana

http://engineering.purdue.edu/LIBNA

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Key Topics

Biochips/Biosensors and Device Fabrication

Cells, DNA, Proteins

Micro-fluidics

Biochip Sensors & Detection Methods Micro-arrays

Lab-on-a-chip Devices

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Dielectrophoresis

Simplest approximation:

[ ]23

0 Re2 RMSCMm EfrF =

( )mp

mp

mpCMe2e

eee,ef

+

= )(

=

p

p2 < m

mp1 > m

Electrode

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Polystyrene beads : p < m negative DEP

Cells : p < m Negative DEPCells : p > m Positive DEP

Dielectrophoresis on Interdigitated

Electrodes

Interdigitated electrodes

on a chip

H. Li and R. Bashir, Sensors and

Actuators, 2002, JMEMS, 2004

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A Dielectrophoretic Filter

Schematic of the device

cross-section

Detection

Chamber

Electrodes

FlowFlow

Beads

and

bacteria

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Forces on a particle in a

micro-fluidic flow

FHD drag

Fsedimentation

FDEPx

FDEPyFHD lift

h

Interdigitated electrodes

Flow

gRF mpsedi )(3

4 3 =

)(6 pmdragHD kRF

= hx

hx 16

whU= U: flow rate in l/min

1. DEP Force

2. Sedimentation Force

3. Hydrodynamic Drag Force:

Assume a parabolic laminar flow profile:

4. Hydrodynamic lifting force

( ) 0

2 1153.0=

x

mliftHD

dx

d

RxRF

Two orders of magnitude smaller

than typical DEP lifting force

Neglected here

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-40 -30 -20 -10 0 10 20 30 40-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

x 10-13 x-component of DEP force at different heights

Horizontal position of the bead,m

x-c

omponentofDEPfo

rce,

N

0.6m

1.6m

2.6m

3.6m

4.6m

X-component of DEP force at different

heights

Bead diameter:

0.7m

Bead conductivity:

2e-4 S/m

Relative permittivity

of bead: 2.6 Bead density: 1.05

g/cm3

Medium (DI water)conductivity: 2.5

S/m

Relative permittivity

of medium: 80

Medium density: 1.0

g/cm3

Voltage: 1Vrms

Frequency: 580KHz

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-40 -30 -20 -10 0 10 20 30 400

0.5

1

1.5

2

2.5

3x 10

-12

Horizontal position of the bead,m

VerticalDEPforce,

N

1.2m

2.2m

3.2m

4.2m

Y-component of DEP force at different

heights

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Trapping of beads (- DEP) and

microorganisms (+ DEP)

0 0.1 0.2 0.3 0.4 0.5 0.6 0.70

50

100

150

200

250

300

Flow rate (l/min)

Voltage

2(V

2p-p)

yeast cells

B. Cereus spores

Listeria innocua bacteria

Vaccinia virus

0 0.02 0.04 0.06 0.08 0.1 0.120

50

100

150

200

250

300

350

Flow rate (l/min)

Voltage

2(V

2p-p)

2.38m bead

5.44m bead

Simulation

Experiments

H. Li, Y. Zheng, D. Akin, R. Bashir,IEEE/ASME JMEMS, 2005

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Dielectrophoretic Trapping ofVaccinia

virus (positive DEP)

Virus Size ~ 250x350nm

Picture taken at: 10Vpp, 1MHz, DI water

~1.5S/cm, flow rate ~0.1l/min

X 375%

400x magnification: viral surface lipid

membrane labeled green (DiOC63) and

viral nucleic acids were stained blue(Hoechst 33342 stain)

D. Akin, H. Li, R. Bashir, Nano Letters, 4, pp. 257 -259, 2004

40m40m

Electrode Edges

Virus particles 10m

The dual (DiOC63, green and DiL, red)

labelled viral particles

Electrode Edges

Virus particles 10m

The dual (DiOC63, green and DiL, red)

labelled viral particles

Fluorescent imaging of nano-scale virusparticles (Vaccinia virus and Human

Corona Virus)

Trapping of viruses in DEP filters

Dual labeling of viruses with fluorescentdyes

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Release voltage vs. diameter for particle collecting the electrode

edge, considering the Brownian motion

Equivalent external force due to

Brownian motion is estimated to

be 20kT/?d 8.210-15 N,

k is Boltzmann constant,

T is the absolute temperature in

Kelvin, and

?d is the trap width, assumed tobe 10m

Polarization factor=0.5,

flow rate 0.1m/min, in thechannel with cross-section350x11.6m2,

interdigitated electrodes

with 23m width and17mspacing

hfEr

kV

hrk

hr

hrkrEfrV

CM

CM

]Re[

18

3636]Re[2

2

2

2

2232

=

=

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Micro-fluidic Characterization

Micro-Particle Imaging Velocimetry (PIV)

Flood IlluminationFlood Illumination

Microscope ObjectiveMicroscope Objective

Glass

DeviceFlow In

Focal PlaneFocal Plane

Flow Out

Wereley, et al. Purdue Gomez, et al. 2001

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Key Topics

Biochips/Biosensors and Device Fabrication

Cells, DNA, Proteins

Micro-fluidics

Biochip Sensors & Detection Methods Micro-arrays

Lab-on-a-chip Devices

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Biochip Sensors

Detect cells (mammalian, plant, etc.),

microorganisms (bacteria, etc.), viruses,

proteins, DNA, small molecules

Use optical, electrical, mechanical

approaches at the micro and nanoscale in

biochip sensors

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Electrical Detection

Conductometric Detection

Z (interface)Z (bulk)

Measurement electrodes

Measurement

Volume

Measurement electrodes

Potentiometric Detection

Source Drain(+ve voltage)

ISFETReferenceelectrode

Current flow

Capture/sensor layer

Reference

electrode

Current flow

Ions oranalytes

(b)

Amperometer DetectionReference

electrode

Working Electrode

Reference

electrode

GlucoseGOD

+ + + +

-

e-

Surface Stress Change Detection

z

L

t

z = deflection of the free end of the cantilever L = cantilever length t = cantilever thickness E = Youngs modulus

= poisons ratio

1change in surface stress on top surface

2 change in surface stress on bottom surface

( )( )

21

21

4

=

Et

lz

Surface Stress Change Detection

z

L

t

z = deflection of the free end of the cantilever L = cantilever length t = cantilever thickness E = Youngs modulus

= poisons ratio

1change in surface stress on top surface

2 change in surface stress on bottom surface

( )( )

21

21

4

=

Et

lz

=

221

2

11

4 off

km

m

kf

2

1=

k = spring constant

m = mass of cantilever

f0= unloaded resonant frequency f1 = loaded resonant frequency

Mass Change Detection

=

221

2

11

4 off

km

m

kf

2

1=

k = spring constant

m = mass of cantilever

f0= unloaded resonant frequency f1 = loaded resonant frequency

Mass Change Detection

Mechanical Detection

(a)

Sensing Methods in BioChips

Optical Detection

(c)

Captureprobes

TargetProbes

Fluorescencedetection

DNA detection on chip surfaces

Capture

probes

Fluorescence

detection

Protein detection on chip surfaces

TargetProbes

Captureprobes

Cell detection on chip surfaces

Captureprobes

TargetProbes

Fluorescencedetection

DNA detection on chip surfaces

Capture

probes

Fluorescence

detection

Protein detection on chip surfaces

TargetProbes

Captureprobes

Cell detection on chip surfaces

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Surface Stress Change Detection

z

L

t

z = deflection of the free end of the cantilever L = cantilever length

t = cantilever thickness

E = Youngs modulus = poisons ratio 1 change in surface stress on top surface

2 change in surface stress on bottom surface

( )( )21

21

4

=

Et

lz

Surface Stress Change Detection

z

L

t

z = deflection of the free end of the cantilever L = cantilever length

t = cantilever thickness

E = Youngs modulus = poisons ratio 1 change in surface stress on top surface

2 change in surface stress on bottom surface

( )( )21

21

4

=

Et

lz

Mechanical Detection

1. Microcantilever Stress Sensors

0.2m thick, 100m long,silicon cantilevers

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IBM Zurich Research:

DNA Detection

Fritz et al, Science, 288,April 2000

Microcantilever Stress Sensors

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Detection of PSA,

Prostate Specific Antigen(cancer marker protein in blood)

Microcantilever Stress Sensors

Wu et al, Nature Biotechnology, 19, September 2001 Deflections have been measured with a resolution of0.4x10-12 m.*

- PSA ~ 30kDa ~ 30 x 1e3 x 1.66e-24gm

- In 1ng/ml ~ 2e10 molecules/ml- Area of 20um x 60um, each protein 10nm x 10nm ~1e8 proteins

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Polymer/Silicon Cantilever Sensors

Environmentally sensitive micro-patterned polymer structures on

cantilevers Hydrogel patterned on cantilever and then exposed to varying pH

50 m

Cantilever

over a well

PMMA-based

Hydrogel

Polymer

R. Bashir, J.Z. Hilt, A. Gupta, O. Elibol, and N.A. Peppas, Applied Physics Letters, Oct 14th, 2002;J. Zachary Hilt, Amit K. Gupta, Rashid Bashir, Nicholas A. Peppas Biomedical Microdevices, September 2003, Volume 5, Issue 3,177-184

- pH = 1-10e-5

- pH = 6.5 ~ 1.9e5 H+ in 1000m3

- pH = 5e-4 change of ~ 150 H+

0

5

10

15

20

25

2.5 3.5 4.5 5.5 6.5 7.5 8.

pH of Fluid Around the Cantilever

Deflectionatthetip(m)

Cantileverstouched the

bottom

ExperimentModel

slope = 18.3mm/pH(=1nm/5e-5pH)

-

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Piezoelectric

Driven

Thermal

NoiseSpectra

Cantilever BeamLength = 10 m

Thickness = 30 nm

f0

= 270 kHz

Mass Change DetectionMass Change Detection

2. Microcantilever Mass Sensors

Unloaded Resonant Frequency :

Spring constant for a rectangular

shaped cantilever beam:

=

m

kf2

10

3

3

4l

wEtk =

= 22

12

114 offkm

k = spring constant

m = mass of cantilever

f0= unloaded resonant frequency

f1 = loaded resonant frequency

= 22

12

114 offkm

k = spring constant

m = mass of cantilever

f0= unloaded resonant frequency

f1 = loaded resonant frequency

Loaded Resonant frequency :

m is the added massmm

kf

+=

2

11

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Detection of Bacterial Mass

Craighead, et al. APL, 77, 3, 17th July 2001, 450-452

o2 =

k

m

E-coli Cells

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Detection of Listeria Cell Mass

Frequency change after binding of 180 dry bacteria

cellsUnloaded

ResonantFrequency

(85.6 kHz)

Loaded

ResonantFrequency

(84.7 kHz)

6.5 7 7.5 8 8.5 9.59 10x 104Frequency (Hz)

Amplitude(units)

10-11

10-10

10-9

10-8

Individual Listeria innocua

bacterial cells10 m1 m

Non-specific binding ofListeria innocua bacterial

cells to a cantilever beam

Lx

m* = (x/L)m

A. Gupta, D. Akin, R. Bashir, JVST-B, 2004.

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kQ

TBkf

A

B

2

1 0

43

4541

k

m

Q

TBk

A

effB

The frequency measurement is limited

by thermo-mechanical noise on the

cantilever beam.

Minimum Detectable Frequency,

? f,min =

Minimum Detectable Mass,?m,min =

kB = Boltzmann constant

T = Temperature in Kelvin

B = Bandwidth measurement, (~ 1 kHz)

Q can increase by 100X by driving the

cantilevers

Minimum Detectable Mass

3 m 4 m 5 m 6 m 7 m 8 m 9m

Length of cantilever beam (m)

Width of cantilever beam = 1 m

Thickness of cantilever beam = 10 nm

10 m3 m 4 m 5 m 6 m 7 m 8 m 9m

Length of cantilever beam (m)

Width of cantilever beam = 1 m

Thickness of cantilever beam = 10 nm

10 m

Roukes, et al.

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Fabrication Process Flow

Silicon

Silicondioxide

PECVD Silicondioxide

Materials Legend (a)

(b)

(e)

Top viewCross-sectional view

(c)Etch Window

(d)Bottom ofchannel

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25A. Gupta, D. Akin, R. Bashir, App l ied Phy sics L etters, March 15, 2004.

SEM Pictures of Cantilevers

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Frequency Shift vs. No. of Particles

- 1 kHz frequency shift for 160 ag

- Sensitivity ~ 6.3 Hz/ag

Average mass of Vaccinia Virus ~ 9.5fg

Work on going to integrated concentration elements

Integrated Abs on cantilevers

f0 = 1.27 MHz

f1 = 1.21 MHz

Q ~ 5

k = 0.006 N/m

?f=60kHz

A. Gupta, D. Akin, R. Bashir, App l ied Phy sics L etters, March 15, 2004.

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0.8 1 1.2 1.4 1.6 1.8 2 2.2

10-10

10-9

Power Spectral Density Plot at Various Stages of Biosensor Analysis

Frequency (in Hz)

Amplitude(units)

Unloaded cantilever beamCantilever beam after absCantilever beam after virus capture

UnloadedResonant

Frequency,1.256 MHz

ResonantFrequency

after AntibodyAttachment,

1.889 MHz

ResonantFrequencyafter VirusCapture,

1.757 MHz

1.5 2 2.5 3 3.510

-11

10-10

10-9

Power Spectral Density Plot at Various Stages of Biosensor Analysis

Frequency (in MHz)

Amplitude(arb.units)

Unloaded Resonant Frequency

Resonant Frequency after Antibody Attachment

Resonant Frequency after Virus Capture

Unloaded

Resonant

Frequency,

2.751 MHz

Resonant

Frequency

after AntibodyAttachment,

2.556 MHz

ResonantFrequency

after Virus

Capture,2.427 MHz

Virus capture experiment: odecreases with Ab attachment and

with antigen capture

BSA and Biotinylated-BSA

Vaccinia virus

Biotinylated antibody

Chip

Streptavidin

Specific Capture of Virus Particles

Virus capture experiment: oincreases with Ab attachment and

decreases with antigen capture

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28Ilic, B., Craighead, H.G.; Krylov, S.; Senaratne, W.; Ober, C.; Neuzil, P.Source: Journal of Applied Physics, v 95, n 7, 1 April 2004, p 3694-703

Mass of Molecules

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Electrical/Electrochemical Detection

1. amperometric biochips, which involves theelectric current associated with the electronsinvolved in redox processes,

2. potentiometric biochips, which measure a

change in potential at electrodes due to ions orchemical reactions at an electrode (such as anion Sensitive FET), and

3. conductometric biochips, which measure

conductance changes associated with changesin the overall ionic medium between the twoelectrodes.

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1. Amperometric Detection

Reference

electrode

Working Electrode

Reference

electrode

GlucoseGOD

+ + + +

-

e-

-D-Glucose + O2 + H2O D-gluconic acid + H2O2

hydrogen peroxide is reduced at -600mV at Ag/AgCl anode reference electrode.

Glucose Oxidase

Perdomo, et al., 2000

Detection of Glucose, Lactate,

Urea, etc.

Enzyme entrapped in a gel

Surface regeneration and

sensor reusability

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31Umek, et al. J. Molecular Diagnostics, 3, 74-84, 2001

Drummond, Hill, Barton, Nature Biotech, v21, n10, Oct 2003, p1192htt ://www.motorola.com/lifesciences/esensor/tech bioelectronics.html

Detection of DNA Hybridization

Capture probes are attached to

electrodes. Target DNA binds to complementary

probes

DNA sequences, called signalingprobes, with electronic labels attachto them (ferrocene-modified DNA

oligonucleotides, E1/2 of 0.120 V vs.Ag/AgCl, act as signaling probes).

Binding of the target sequence toboth the capture probe and thesignaling probe connects theelectronic labels to the surface.

The labels transfer electrons to theelectrode surface, producing acharacteristic signal.

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2. Potentiometric Sensors

ISFETs, ChemFETs, etc.

Potential difference between the gate and the reference electrode in

the solution

Change in potential converted to a change in current by a FET or toa change in capacitance in low doped silicon

Gate material is sensitive to specific targets

pH, Ions, Charges

Source Drain

(+ve voltage)

ISFETReference

electrode

Current flow

Capture/sensor layer

Reference

electrode

Current flow

Ions oranalytes

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Nanoscale pH Sensors

Label Free !!

Detection of pH change

Detection of protein binding

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Integrated Silicon Nanowire Sensors

Objectives:

- Bio-sensors with electronic output

- Capability of dense arrays integrated

with ULSI silicon

- Direct Label Free Detection of DNA and

Proteins

Plate Size ~ 20nm X 1m X 3m

Wire Size ~ 20nm X 20nm X 3m

P type Silicon

oxide

Metal N+N+

N-/P-

Integrated

Bottom Gate

Capture

Molecules

Target

Molecules

Electrical response of the device uponexposure to oxygen (red dotted lines) and

nitrogen (blue solid lines)

O. H. Elibol, D. Morisette, D. Akin, R. Bashir, Applied Physics

Letters. Volume 83, Issue 22, pp. 4613-4615, December 1, 2003

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Field Effect Sensing of DNA

J. Fritz, Emily B. Cooper, Suzanne Gaudet, Peter K. Sorger, and Scott R. Manalis, Electronic detection of

DNA by its intrinsic molecular charge, PNAS 2002 99: 14142-14146.

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Nanoparticle Mediated DNA Detection

Au nanoparticles assemble between two electrodes if DNA is

hybridized

Silver staining of the Au nanoparticles

Conductance changes between micro-scale electrodes indicate

DNA hybridization

Sensitivity of 5x10-13 M shown

Park S J ; Taton T A ; Mirkin C A Array Based Electrical Detection